METHOD AND APPLICATION OF UNSYMMETRICALLY meso-SUBSTITUTED PORPHYRINS AND CHLORINS FOR PDT

ABSTRACT

Biologically active compounds are provided that can be used as photosensitizers for diagnostic and therapeutic applications, particularly for PDT of cancer, infections and other hyperproliferative diseases, fluorescence diagnosis and PDT treatment of a non-tumorous indication such as arthritis, inflammatory diseases, viral or bacterial infections, dermatological, ophthalmological or urological disorders as well as providing methods to obtain them in pharmaceutical quality. One embodiment consists of a method to synthesize a porphyrin with a defined arrangement of meso-substituents and then converting this porphyrin system to a chlorin system by dihydroxylation or reduction, and if more than one isomer is formed separate them by chromatography either on normal or reversed phase silica. In another embodiment the substituents on the porphyrin are selected to direct the reduction or dihydroxylation to the chlorin so that a certain isomer is selectively formed. Another embodiment is to provide amphiphilic compounds with a higher membrane affinity and increased PDT-efficacy. In another embodiment a method to reductively cleave the osmate(VI)ester avoiding the use of gaseous H 2 S is provided. In another embodiment substituents are identified that via their steric and/or electronic influence direct the dihydroxylation or reduction with diimine so that one isomer is favored. Another embodiment consists of formulate the desired isomer into a liposomal formulation to be injected avoiding undesirable effects like solubility problems or delayed pharmacokinetics of the tetrapyrrole systems.

NATIONAL FILING UNDER 35 USC 371

This application is being filed as a US National stage under 35 USC 371of PCT Application No. PCT/US09/57283, which was filed Sep. 17, 2009 andalso claims the benefit of U.S. Provisional Application Ser. No.61/098,026 filed Sep. 18, 2008, both of which are incorporated byreference herein.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to the chemistry of biologically active compounds.More particularly to specifically substituted porphyrin and chlorinderivatives that can be used as photosensitizers for a wide range oflight irradiation treatments such as photodynamic therapy of cancer,infections and other diseases.

2. Invention Disclosure Statement

Photodynamic therapy (PDT) is one of the most promising new techniquesnow being explored for use in a variety of medical applications(Photodynamic therapy, basic principles and clinical applications. Eds.B. W. Henderson, Th. J. Dougherty, Marcel Dekker, 1992, New York), andparticularly is a well-recognized treatment for the destruction oftumors (Photodynamic tumor therapy. 2^(nd) and 3^(rd) generationphotosensitizers. Ed. J. G. Moser, Harwood Academic Publishers, 1998,Amsterdam). Photodynamic therapy uses light and a photosensitizer (adye) to achieve its desired medical effect. A large number of naturallyoccurring and synthetic dyes have been evaluated as potentialphotosensitizers for photodynamic therapy. Perhaps the most widelystudied class of photosensitizers are the tetrapyrrolic macrocycliccompounds. Among them, especially porphyrins and chlorins have beentested for their PDT efficacy. Porphyrins are macrocyclic compounds withbridges of one carbon atom joining pyrroles to form a characteristictetrapyrrole ring structure. There are many different classes ofporphyrin derivatives including those containing dihydro-pyrrole units.Chlorins, as referred to in the present invention, are porphyrinderivatives containing one dihydro-unit whereas bacteriochlorins arecharacterized by two dihydro-pyrrole units (in general in chlorins onedouble bond of the aromatic system in β-position is absent and inbacteriochlorins two opposite double bonds are absent compared to theporphyrin). As examples of tetrapyrrolic macrocyclic compounds used asphotosensitizers, U.S. Pat. No. 4,656,186 from Bommer et. al. disclosesfluorescent mono, di- or polyamide of an aminocarboxilic acid andtetrapyrrole containing at least three carboxy groups, U.S. Pat. No.7,022,843B1 from MacAlpine et. al. provides β,β′-dihydroxymeso-substituted chlorin as photosensitizers, and U.S. Pat. No.7,166,719B2 from Pandey et. al. discloses tetrapyrrole compoundscontaining a fluorinated substituent where the compound is a chlorin ora bacteriochlorin for PDT diagnostic and therapeutic application.

There are several properties that an effective photosensitizer shouldaccomplish. Among them, a desirable characteristic in order toefficiently destroy deep target tissues is a strong absorption at longwavelength. Many current photosensitizers are not efficient enough asthey have low absorption in the red region of the spectrum. Chlorinshave the advantage that they possess an intense absorption in the redand near-infrared region of the electromagnetic spectrum. As light oflonger wavelength penetrates deeper into the tissue, it is thus possibleto treat e.g. more expanded tumors, if the PDT is employed for tumortherapy. Chlorins possessing potential for PDT can either be derivedfrom natural sources or from total synthesis.

If the chlorins are derived from natural compounds they are usuallyobtained by derivatizing chlorophylls or bacteriochlorophylls, as forexample the photosensitizers derived from chlorophyll a ofphotosynthetic plants and algae disclosed in U.S. Pat. No. 5,330,741.Due to the sensibility of the natural compounds this is often difficultand requires vast resources. So, the synthesis of chlorins by totalsynthesis is an appealing alternative. Methods to prepare chlorins andbacteriochlorins by total synthesis are known in the art. Generallythese compounds are prepared by first synthesizing the porphyrin andthen converting the porphyrin system to a chlorin or bacteriochlorinsystem. This step can e.g. be performed by the reduction with in situgenerated di-imine or by cis-dihydroxylation with osmium tetroxide;multistep reactions leading to trans-dihydroxylation are also known(patent EP 00337601B1; patent application WO 09613504A1, patentapplication WO 00061584A1; C. Bruckner, D. Dolphin,2,3-vic-Dihydroxy-meso-tetraphenylchlorins from the Osmium TetroxideOxidation of meso-Tetraphenylporphyrin, Tetrahedron Lett. 1995, 36,3295-3298; C. Brückner, D. Dolphin, β,β′-Dihydroxylation ofmeso-Tetraphenylchlorins, Tetrahedron Lett. 1995, 36, 9425-9428; H. W.Daniell, S. C. Williams, H. A. Jenkins, C. Brückner, Oxidation ofmeso-tetra-phenyl-2,3-dihydroxychlorin: simplified synthesis ofβ,β′-dioxochlorins, Tetrahedron Lett. 2003, 44, 4045-4049; F. Rancan, A.Wiehe, M. Nöbel, M. O. Senge, S. Al Omani, F. Böhm, M. John, B. Röder,influence of substitutions on asymmetric dihydroxychlorins with regardto intracellular uptake, sub cellular localization andphotosensitization in Jurkat cells, J. Photochem. Photobiol. B: Biology2005, 78, 17-28; I. Laville, T. Figueiredo, B. Loock, S. Pigaglio, Ph.Maillard, D. S. Grierson, D. Carrez, A. Croisy, J. Blais, Synthesis,Cellular Internalization and Photodynamic Activity of Gluco-conjugatedDerivatives of Tri and Tetra(meta-hydroxyphenyl)chlorines, Bioorg. Med.Chem. 2003, 11, 1643-1652). Mostly, compounds with four identicalsubstituents in the meso-positions have been investigated and tested fortheir PDT efficacy. One prominent example is Temoporfin which is theactive compound in the medicinal product Foscan® which is successfullyused in Europe as a medicinal product for the PDT treatment of head andneck cancer. Also, all examples in the abovementioned patent applicationWO 09613504A1 are compounds with four identical meso substituents. Thefew publications on unsymmetrically tetrakis-meso-substituted chlorinsderived from total synthesis that exist are of the so-called A₃B-type,i.e. incorporating 3 identical and one different meso-substituent (I.Laville, T. Figueiredo, B. Loock, S. Pigaglio, Ph. Maillard, D. S.Grierson, D. Carrez, A. Croisy, J. Blais, Synthesis, CellularInternalization and Photodynamic Activity of Glucoconjugated Derivativesof Tri and Tetra(meta-hydroxyphenyl)chlorines, Bioorg. Med. Chem. 2003,11, 1643-1652, F. Rancan, A. Wiehe, M. Nöbel, M. O. Senge, S. Al Omani,F. Böhm, M. John, B. Röder, Influence of substitutions on asymmetricdihydroxychlorins with regard to intracellular uptake, sub cellularlocalization and photosensitization in Jurkat cells. J. Photochem.Photobiol. B: Biology 2005, 78, 17-28; J. K. Macalpine, R. Boch, D.Dolphin, Evaluation of tetraphenyl-2,3-dihydroxychlorins as potentialphotosensitizers, J. Porphyrins Phthalocyanines 2002, 6, 146-155). Onereason for using symmetrically substituted porphyrins to convert theminto chlorins is that in this case no isomers are formed. If no isomersare formed the resulting compounds are easily characterized andprepared, a key factor for commercial production. If unsymmetricallysubstituted porphyrins are used to convert them into chlorins differentregioisomers are formed which require subsequent separation (not in thecase of a trans-arrangement of the substituents, cf. FIG. 2). Therefore,the chlorins with a meso-A₃B-substitution found in the art are oftenpoorly characterized or are used as an isomeric mixture withoutseparation (e.g. J. K. Macalpine, R. Bach, D. Dolphin, Evaluation oftetraphenyl-2,3-dihydroxychlorins as potential photosensitizers, J.Porphyrins Phthalocyanines 2002, 6, 146-155; I. Laville, T. Figueiredo,B. Loock, S. Pigaglio, Ph. Maillard, D. S. Grierson, D. Carrez, A.Croisy, J. Blais, Synthesis, Cellular Internalization and PhotodynamicActivity of Glucoconjugated Derivatives of Tri andTetra(meta-hydroxyphenyl)chlorines, Bioorg. Med. Chem. 2003, 11,1643-1652). As it is difficult to purify the mixture in order toeliminate the isomers that do not contribute to the PDT effect or enrichthe preparation with active compounds, it would be an advantage to findalternative unsymmetrically tetrakis-meso-substituted chlorins easilycharacterized and produced with simple preparation methods. Especiallyto seize the particular properties of unsymmetrically substitutedchlorins, as they might increase the amphiphilicity of the compounds andthus their membrane affinity and PDT efficacy.

Thus, there is a need to enhance the effectiveness of prior artbiologically active compounds used as photosensitizers in order tosuccessfully perform a wide range of light irradiation treatments suchas photodynamic therapy of cancer, infections and other diseases.Moreover, it is necessary to provide novel methods of preparation andapplication of unsymmetrically tetrakis-meso-substituted chlorins inorder to provide enhanced photosensitizers than those available up todate. Thus, PDT efficacy would be increased by taking advantage ofunsymmetrically tetrakis-meso-substituted chlorins properties, such asstrong absorption at long wavelength of the red and near-infrared regionof the electromagnetic spectrum for deeper tissue penetration, enhancedselectivity for tumors or other target tissues over healthy surroundingtissues due to its tailored amphiphilicity that increases membraneaffinity, and custom-made pharmacokinetic behavior depending on theparticular PDT application.

OBJECTIVES AND BRIEF SUMMARY OF THE INVENTION

It is an objective of the present invention to provide biologicallyactive compounds that can be used as photosensitizers for a wide rangeof applications including light irradiation treatments such asphotodynamic therapy of cancer, infections and other diseases.

It is a further objective of the present invention to use the chemicallystable porphyrin and chlorin derivatives for various medicalapplications such as photodynamic therapy.

It is another objective of the present invention to provideunsymmetrically tetrakis-meso-substituted chlorin structures that can beused in the photodynamic therapy of tumors and other hyperproliferativediseases, dermatological disorders, viral or bacterial infections,ophthalmological disorders or urological disorders.

It is yet another object of the present invention to provideunsymmetrically tetrakis-meso-substituted chlorin structures that can beused for the fluorescence diagnosis and PDT treatment of a non-tumorousindication such as arthritis and similar inflammatory diseases.

It is still another object of the present invention to provide a methodto prepare and purify such unsymmetrically tetrakis-meso-substitutedchlorins and to provide a method for the separation of the isomersformed.

It is still a further object of the present invention to provide highlyamphiphilic compounds to be used in the PDT-treatment of tumors,dermatological disorders, viral or bacterial infections,ophthalmological disorders or urological disorders.

It is another object of the present invention to provide a method ofpreparation that can direct the dihydroxylation or reduction of thestarting material so that the formation of one isomer is favored.

It is still another objective to provide pharmaceutically acceptableformulations for the biologically active compounds of the presentinvention such as liposomal formulation to be injected avoidingundesirable effects like precipitation at the injection site or delayedpharmacokinetics of the tetrapyrrole systems.

Briefly stated, the present invention provides Biologically activecompounds that can be used as photosensitizers for diagnostic andtherapeutic applications, particularly for PDT of cancer, infections andother hyperproliferative diseases, fluorescence diagnosis and PDTtreatment of a non-tumorous indication such as arthritis, inflammatorydiseases, viral or bacterial infections, dermatological,ophthalmological or urological disorders as well as providing methods toobtain them in pharmaceutical quality. One embodiment consists of amethod to synthesize a porphyrin with a defined arrangement ofmeso-substituents and then converting this porphyrin system to a chlorinsystem by dihydroxylation or reduction, and if more than one isomer isformed separate them by chromatography either on normal or reversedphase silica. In another embodiment the substituents on the porphyrinare selected to direct the reduction or dihydroxylation to the chlorinso that a certain isomer is selectively formed. Another embodiment is toprovide amphiphilic compounds with a higher membrane affinity andincreased PDT-efficacy. In another embodiment a method to reductivelycleave the osmate(VI)ester avoiding the use of gaseous H₂S is provided.In another embodiment substituents are identified that via their stericand/or electronic influence direct the dihydroxylation or reduction withdiimine so that one isomer is favored. Another embodiment consists offormulating the desired isomer into a liposomal formulation to beinjected avoiding undesirable effects like solubility problems atinjection or delayed pharmacokinetics of the tetrapyrrole systems

The above and other objects, features and advantages of the presentinvention will become apparent from the following description read inconjunction with the accompanying drawings.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 shows examples of unsymmetrically tetrakis-meso-substitutedchlorin structures combining two nonpolar (alkyl) and two polarmeso-substituents that are specially suited for medical applications.

FIG. 2 depicts an embodiment of the present invention showing thespecifically substituted porphyrin derivatives, particularly the chlorinderivatives of types 1, 2, 3 or 4.

FIG. 3 shows an embodiment of the present invention depicting thearrangement of a porphyrin system to be converted to a chlorin system,where the porphyrin system is of the A₂B₂ type either with a “cis” or a“trans” arrangement of the meso-substituents and A is the nonpolar(alkyl) and B the polar substituent.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention provides biologically active compounds that can beused as photosensitizers for a wide range of light irradiationtreatments such as photodynamic therapy of cancer, hyperproliferativediseases, dermatological disorders, viral or bacterial infectiousdiseases, ophthalmological disorders and/or urological disorders. Thealternative photosensitizers provided by the present invention have theadvantage that they are easily produced and characterized. Moreover, asthe present invention provides methods to tailor amphiphilic compoundsfor desired PDT applications, target tissue selectivity is increased andthus PDT efficacy. The present invention enhances the effectiveness ofprior art biologically active compounds offering a deeper tissuepenetration due to their strong absorption at long wavelength of the redand near-infrared region of the electromagnetic spectrum, enhancedselectivity for target tissues over healthy surrounding tissues due toits tailored amphiphilicity and custom-made pharmacokinetic behaviordepending on the particular PDT application.

The biologically active compounds of the present invention that can beused for different medical indications, particularly PDT, areunsymmetrically tetrakis-meso-substituted chlorin structures. Indeed, ithas been unexpectedly been found that chlorins combining two nonpolar(alkyl) and two polar meso-substituents in their structure, asillustrated in FIG. 1, are especially suited for such a medicalapplication. Additionally, the novel invention extends its applicationsas it can be used for fluorescence diagnosis and PDT treatment of anon-tumorous indication such as arthritis and similar inflammatorydiseases.

In order to obtain the novel photosensitizers the present invention usesthe chemically stable porphyrin and chlorin derivatives according toformulae 1, 2, 3, and 4 shown in FIG. 2 and provides methods ofpreparation and separation of the isomers formed to obtain meso-alkylsubstituted chlorins, more particularly the unsymmetricallytetrakis-meso-substituted chlorin structures that can be used in thephotodynamic therapy. With respect to partially meso-alkyl substitutedchlorins there is in fact only one example in the literature (F. Rancan,A. Wiehe, M. Nöbel, M. O. Senge, S. Al Omani. F. Böhm, M. John, B.Röder, Influence of substitutions on asymmetric dihydroxychlorins withregard to intracellular uptake, sub cellular localization andphotosensitization in Jurkat cells, J. Photochem. Photobiol. B: Biology2005, 78, 17-28; the compound is also of the meso-A₃B-substitutionpattern). On the other hand, especially such unsymmetrically substitutedchlorins, which are regio-isomerically pure (though in most cases thereare still enantiomeric mixtures), could be of great interest asphotosensitizers for PDT as such unsymmetric substitution might increasethe amphiphilicity of the compounds and thus their membrane affinity andPDT efficacy. In addition, it has surprisingly been found during theinvestigations related to the present invention, that there aresometimes pronounced differences in PDT-efficacy between differentchlorin isomers.

An embodiment of the present invention consists of a method tosynthesize a porphyrin with a defined arrangement of meso-substituents[a porphyrin of the A₂B₂ type, either with a ‘cis’ or a ‘trans’arrangement of the meso-substituents, as illustrated in FIG. 3, wheree.g. A is the nonpolar (alkyl) and B the polar substituent] and thenconverting this porphyrin system to a chlorin system by dihydroxylationor reduction (as e.g. described in: M. Schroeder, Osmium Tetraoxide CisDihydroxylation of Unsaturated Substrates, Chem. Rev. 1980, 80, 187-213;R. Bonnett, R. D. White, U.-J. Winfield, M. C. Berenbaum,Hydroporphyrins of the meso-tetra(hydroxyphenyl)porphyrin series astumor photosensitizers, Biochem. J. 1989, 261, 277-280). In a last stepthe isomers (if more than one isomer is formed) are separated bychromatography either on normal or reversed phase silica.

Another embodiment of the present invention consists of the steps ofsynthesizing a porphyrin with a defined arrangement of substituents,converting it to the chlorin, separating the isomers as described aboveand then to formulate the desired isomer into a liposomal formulation.

In yet another embodiment of the present invention a porphyrin of the‘trans’-A₂B₂-type is synthesized, converted to the dihydroxychlorin andpurified by chromatography.

In yet another embodiment of the present invention, a porphyrin of the‘cis’-A₂B₂-type is synthesized, converted to the dihydroxychlorin andthen the isomers are separated and purified by chromatography.

It has also been found that the substituents on the porphyrin due totheir electronic and steric influence can direct the dihydroxylation,thus favoring the formation of one isomer. So, in yet another embodimentof the present invention the substituents on the porphyrin are selectedto direct the reduction or dihydroxylation to the chlorin (examples 3.2and 3.4) so that a certain isomer is selectively formed.

In a specifically preferred embodiment of the present invention aporphyrin of the ‘trans’-A₂B₂-type is synthesized, having hexyl chainsas substituent A and methoxycarbonyl phenyl residues as substituent B.Then this porphyrin is converted to the dihydroxychlorin and theremaining methylester is hydrolyzed to receive the correspondingcarboxylic acid.

Acceptable starting materials for the synthesis of the chlorins whichare the subject of the present invention are pyrrole and aldehydes. Morespecifically, pyrrole and two aldehydes, one alkanal and one aromaticaldehyde are employed for the synthesis of the unsymmetricallysubstituted porphyrins which are the basis of the synthesis of thecorresponding chlorins. Pyrrole and aldehydes are subjected to acondensation reaction. Suitable methods for this condensation have longbeen known in the art (J. S. Lindsey, I. C. Schreiman, H. C. Hsu, P. C.Kearney and A. M. Marguerettaz, J. Org. Chem. 1987, 52, 827-836).Alternatively, the unsymmetrically substituted porphyrins can also besynthesized using di- or tripyrromethanes and aldehydes, as is alsoknown in the art (C.-H. Lee, J. S. Lindsey, One-Flask Synthesis ofMeso-Substituted Dipyrromethanes and Their Application in the Synthesisof Trans-Substituted Porphyrin Building Blocks, Tetrahedron 1994, 50,11427-11440). After condensation and purification of the desiredunsymmetrically substituted porphyrins these are converted to thechlorins. As there is only one example of a tetra-meso-substitutedchlorin bearing only one alkyl-substituent known in the art, anotherembodiment of the present invention provides a method for thepreparation of multiply meso-alkyl-substituted chlorins viadihydroxylation. The synthesis of meso-substituted chlorins bearingalkyl chains is exemplified with examples 3.1-3.4. Furthermore, the useof lipophilic alkyl-substituted porphyrins as substrates for thedihydroxylation is a key feature of the present invention as it givesaccess to amphiphilic compounds with a higher membrane affinity andincreased PDT-efficacy. In examples 1.1 and 1.2 a series ofunsymmetrically substituted porphyrins is synthesized with the objectiveto obtain porphyrins bearing both hydrophilic and hydrophobic groups.

The dihydroxylation of porphyrins with osmium tetroxide that is known inthe art (cf. above Brückner, et al.) uses gaseous H₂S to reductivelycleave the osmate(VI)ester. The use of gaseous and toxic H₂S is notfavorable for the synthesis of compounds to be used eventually in largescale pharmaceutical preparations. Moreover, the use of hydrogen sulfideleads to impurities, making the chromatographic workup and theseparation of the chlorin isomers difficult. Thus, another embodiment ofthe present invention provides a simple method for the reductivecleavage of the osmate(VI)ester that avoids the use of gaseous H₂S.Instead, a small amount of a saturated sodium bisulfite solution inwater/methanol is used which is added to the reaction mixture. Afterstirring the mixture overnight the cleavage of the osmate ester to thediol proceeds quantitatively (examples 3.1-3.4). The resulting chlorinmixtures can easily be separated and purified by chromatography.

As the attack of osmium tetroxide or of the diimine can take place onany of the pyrrolic subunits, the reaction of the unsymmetricallysubstituted porphyrins in the case of the ‘cis’-A₂B₂-type porphyrinsleads to the formation of 3 regioisomers, whereas for the‘trans’-A₂B₂-type only one regioisomer is formed. Therefore, in anotherembodiment the present invention identifies substituents that via theirsteric and/or electronic influence direct the dihydroxylation orreduction with diimine so that one isomer is favored. In the course ofthe investigations related to the present invention it turned out thatfor ‘cis’-A₂B₂-type porphyrins (with A=hexyl) the pyrrolic subunitbetween hexyl-groups (example 3.2 and 3.4) is preferred fordihydroxylation. This degree of selectivity may be caused by simplesteric and/or electronic effects. The structure of the differentregioisomers was unequivocally determined by 2D-NMR-spectroscopy (COSY,HMQC and HMBC). Already the ¹H NMR spectra show the influence of thedihydroxylated pyrrolic subunit on the nearby groups in themeso-positions. Interestingly, as the dipole moment of the chlorins isaffected by the position of the diol also the chromatographic behaviorof the compounds in most cases reflects the structure of thecorresponding regioisomers.

The use of specifically substituted amphiphilic porphyrin and chlorinderivatives produced according to the present invention is suitable tobe used for photodynamic therapy of cancer and other hyperproliferativediseases and infections. In another embodiment, aimed to obtain suchamphiphilic compounds, the methyl ester groups of some porphyrins anddihydroxychlorins were hydrolyzed under basic conditions to provide thecorresponding carboxylic acids (example 2 and 4). These acids have anincreased solubility in polar solvents, increasing their potential asphotosensitizers.

PDT is accomplished by first incorporating the derivatives into apharmaceutically acceptable application vehicle (e.g. ethanolic solutionor liposomal formulation) for delivery of the derivatives to a specifictreatment site. After administering the derivatives in the vehicle to atreatment area, sufficient time is allowed so that the porphyrin andchlorine derivatives preferentially accumulate in the diseased tissue.Lastly, the treatment area is irradiated with light of a properwavelength and sufficient power to activate the porphyrin derivatives toinduce necrosis or apoptosis in the cells of said diseased tissue. Thus,one of the main advantages is that convenient pharmaceuticalformulations can be created for the biologically active compounds of thepresent invention such as liposomal formulation to be injected avoidingundesirable effects like precipitation at the injection site or delayedpharmacokinetics of the tetrapyrrole systems. Due to their amphiphilicnature, the chemically stable porphyrin and chlorin derivatives of thepresent invention can be prepared in various pharmaceutically acceptableand active preparations for different administration methods, e.g.injections. In a specifically preferred embodiment such amphiphiliccompounds are formulated into liposomes (example 8.1 and 8.2). Thisliposomal formulation can then be injected avoiding undesirable effectssuch as precipitation at the injection site or delayed pharmacokineticsof the tetrapyrrole systems.

Determination of dark toxicity (DT) and photo toxicity (example 6.1) ofone specific chlorin derivative of the present invention,5,15-bis-(4-carboxyphenyl)-10,20-dihexyl-7,8-dihydroxy-7,8-chlorinprepared according to example 4.1, in cell culture experiments with a HT29 cell line showed the excellent properties of the compounds for use inPDT. Further examples of the good phototoxic properties of the compoundsof the present invention are illustrated with examples 6.2 and 6.3. Theadditional examples 6.4-6.6 of experiments in the HT 29 cell line areincluded to illustrate that other dihydroxychlorins which do not possessa combination and arrangement of substituents as the one favored in thepresent invention show a less promising PDT activity.

As another object of the present invention is to use the disclosedporphyrin and chlorin derivatives in the diagnosis and treatment ofarthritis and similar inflammatory diseases, the data presented inexamples 7.1-7.4 summarizes the results of the photodynamic treatment oftwo cell lines especially relevant for arthritis (HIG82 and J774A.1, arabbit synoviocyte and a mouse macrophage cell line) with a series ofcompounds of the present invention. Again, a negative example (7.5) of acompound not having the favored combination of substituents and lackingthe photodynamic activity is also included.

The following examples are presented to provide those of ordinary skillin the art with a full and illustrative disclosure and description ofhow to make the chlorin derivatives of the invention and show theirphotodynamic activity and are not intended to limit the scope of whatthe inventor regards as the invention. Efforts have been made to ensureaccuracy with respect to numbers used (e.g. amounts, temperature etc.),but some experimental errors and deviations should be accounted for.Also, best measures have been taken to name the compounds with theirsystematic IUPAC name, nevertheless the basic reference are the givenstructural formulas based on the experimental spectroscopic data.

EXAMPLES

All reagents were used as purchased from commercial suppliers.Tetraacetyl-β-D-glucopyranosyloxy-benzaldehyde (I. Laville, S. Pigaglio,J.-C. Blais B. Loock, Ph. Maillard, D. S. Grieson, J. Blais, Biorg. Med.Chem. 2004, 12, 3673-3682) and5-(4-methoxycarbonylphenyl)-dipyrromethane (B. J. Littler, M. A. Miller,C.-H. Hung, R. W. Wagner, D. F. O'Shea, P. D. Boyle, J. S. Lindsey, J.Org. Chem. 1999, 64, 1391-1396) were prepared according to theliterature. Dichloromethane was purified by distillation over K₂CO₃prior to use. Thin layer chromatography (TLC) was performed using Mercksilica gel 60 (without fluorescence indicator) pre-coated on aluminiumsheets. Flash chromatography was carried out using Merck silica gel 60,0.040-0.063 mm (230-400 mesh). ¹H and ¹³C NMR spectra were recorded inCDCl₃, (CD₃)₂CO or (CD₃)₂SO on Bruker AC 250, AC 500 or AMX 500instruments. Chemical shifts δ are given in ppm relative to TMS asinternal standard or relative to the resonance of the residual solventpeak, J values are given in Hz. Mass spectra were recorded on Varian MAT771, Varian IonSpec QFT-7 or Agilent 6210 ESI-TOF instruments.Electronic absorption spectra were recorded on a Specord S300 (AnalytikJena) spectrophotometer using dichloromethane or acetone as solvent.

Example 1 Preparation of Unsymmetrical Substituted Porphyrins 1.1Preparation of5,15-dihexyl-10,20-bis-(4-methoxycarbonylphenyl)-porphyrin (method A)and 5,10-dihexyl-15,20-bis-(4-methoxycarbonylphenyl)-porphyrin

In a typical experiment, dry dichloromethane (1500 ml) was placed in athree-necked flask equipped with a magnetic stirrer and argon gas inlet.After pyrrole (1.05 ml, 15 mmol), heptanal (1.05 ml, 7.5 mmol) andmethyl 4-formylbenzoate (1.23 g, 7.5 mmol) were added, the flask wasshielded from ambient light and TFA (1.16 ml, 15 mmol) was added and thereaction mixture was stirred at room temperature for 18 h. Then, DDQ(2.55 g, 11.25 mmol) suspended in dry dichloromethane (100 ml) wasadded. After further stirring for 1 h, triethylamine (3 ml) was added.To remove polymeric by-products, the reaction mixture was filteredthrough silica gel. The solvent was evaporated and separation wasachieved via flash chromatography with dichloromethane and furtherpurification with dichloromethane/hexane 3:1 (first band) anddichloromethane (second and third band) as eluent. Further purificationwas achieved by recrystallization from dichloromethane/methanol Thefirst band from the column contained5,10,15-trihexyl-20-(4-methoxycarbonylphenyl)-porphyrin (203 mg, 12%),the second band the title compound5,15-dihexyl-10,20-bis-(4-methoxycarbonylphenyl)-porphyrin (109 mg, 4%)and the third band the title compound5,10-dihexyl-15,20-bis-(4-methoxycarbonylphenyl)-porphyrin (199 mg, 7%).

5,15-Dihexyl-10,20-bis-(4-methoxycarbonylphenyl)-porphyrin

violet microcrystalline solid, mp 238° C.; λ_(max)(CH₂Cl₂)/nm 421 (ε/dm³mol⁻¹ cm⁻¹ 253500), 517 (13400), 553 (7600), 594 (3900) and 650 (5000);δ_(H)(250 MHz; CDCl₃) −2.71 (2H, s, NH), 0.92 (6H, t, J 7.3, 2×CH₃),1.29-1.53 (8H, m, 4×CH₂), 1.76 (4H, m_(c), 2×CH₂), 2.48 (4H, m_(c),2×CH₂), 4.14 (6H, s, 2×OCH₃), 4.89 (4H, t, J 7.7 , 2×CH₂), 8.27 (4H, d,J 8.2, Ar), 8.45 (4H, d, J 8.2, Ar), 8.79 (4H, d, J 5.0, β-H), 9.40 (4H,d, J 5.0, β-H); δ_(C)(63 MHz; CDCl₃) 14.26 (CH₃), 22.84 (CH₂), 30.37(CH₂), 32.04 (CH₂), 35.49 (CH₂), 38.89 (CH₂), 52.55 (OCH₃), 117.88(meso-C), 120.48 (meso-C), 127.93 (Ar), 128.20 (Ar), 129.70 (Ar), 131.46(Ar), 134.61 (Ar), 147.67 (Ar), 167.53 (CO₂CH₃); m/z (ESI) 747.3904([M+H]⁺, C₄₈H₅₁N₄O₄ ⁺ requires 747.3905).

5,10-Dihexyl-15,20-bis-(4-methoxycarbonylphenyl)-porphyrin

violet microcrystalline solid, mp 131° C.; λ_(max)(CH₂Cl₂)/nm 420 (ε/dm³mol⁻¹ cm⁻¹ 263300), 519 (12000), 553 (7000), 596 (3900) and 652 (4000);δ_(H)(250 MHz; CDCl₃)-2.72 (2H, s, NH), 0.95 (6H, t, J 7.3, 2×CH₃), 1.45(8H, m_(e), 4×CH₂), 1.79 (4H, m_(c), 2×CH₂), 2.52 (4H, m_(c), 2×CH₂),4.12 (6H, s, 2×OCH₃), 4.91 (4H, t, J 8.2, 2×CH₂), 8.24 (4H, d, J 8.2,Ar), 8.43 (4H, d, J 8.2, Ar), 8.69 (2H, s, β-H), 8.78 (2H, d, J 5.0,β-H), 9.41 (2H, d, J 5.0, β-H), 9.50 (2H, s, β-H); δ_(C)(63 MHz; CDCl₃)14.30 (CH₃), 22.89 (CH₂), 30.43 (CH₂), 32.06 (CH₂), 35.86 (CH₂), 39.06(CH₂), 52.53 (OCH₃), 117.49 (meso-C), 120.98 (meso-C), 128.05 (Ar),128.63 (Ar), 129.68 (Ar), 130.88 (Ar), 134.63 (Ar), 147.30 (Ar), 167.51(CO₂CH₃); m/z (ESI) 747.3932 ([M+H]⁺, C₄₈H₅₁N₄O₄ ⁺ requires 747.3905).

1.2 Preparation of5,15-dihexyl-10,20-bis-(4-methoxycarbonylphenyl)-porphyrin (method B)

In a typical experiment, acetonitrile (500 ml) was placed in athree-necked flask equipped with a magnetic stirrer and argon gas inlet.After 5-(4-methoxycarbonylphenyl)-dipyrromethane (714 mg, 2.6 mmol) andheptanal (0.36 ml, 2.6 mmol) were added, the flask was shielded fromambient light and TFA (0.2 ml, 2.6 mmol) was added and the reactionmixture was stirred at room temperature for 18 h. Then, DDQ (860 mg, 3.8mmol) suspended in acetonitrile (30 ml) was added. After furtherstirring for 1 h, triethylamine (1 ml) was added. The solvent wasevaporated and preliminary purification was achieved via flashchromatography with dichloromethane/methanol 95:5 as eluent and furtherpurification via flash chromatography with dichloromethane/ethylacetate99:1 as eluent. The title compound5,15-dihexyl-10,20-bis-(4-methoxycarbonylphenyl)-porphyrin was obtainedafter recrystallization from dichloromethane/methanol (81 mg, 9%).

1.3 Preparation of5,15-bis-(3-hydroxyphenyl)-10,20-bis-(tridecyl)-porphyrin and5,10-bis-(3-hydroxyphenyl)-15,20-bis-(tridecyl)-porphyrin

In a typical experiment, dry dichloromethane (1500 ml) was placed in athree-necked flask equipped with a magnetic stirrer and argon gas inlet.After pyrrole (1.05 ml, 15 mmol), tetradecanal (1593 mg, 7.5 mmol) and3-hydroxybenzaldehyde (916 mg, 7.5 mmol) were added, the flask wasshielded from ambient light and TFA (1.16 ml, 15 mmol) was added and thereaction mixture was stirred at room temperature for 4 h. Then, DDQ(2.55 g, 11.25 mmol) suspended in dry dichloromethane (100 ml) wasadded. After further stirring for 1 h, triethylamine (6 ml) was added.To remove polymeric by-products, the reaction mixture was filteredthrough silica gel. The solvent was evaporated and separation wasachieved via repeated flash chromatography withdichloromethane/ethylacetate 90:10 and 95:5 as eluent. Furtherpurification was achieved by recrystallization fromdichloromethane/aqueous methanol The first band from the columncontained 5-(3-hydroxyphenyl)-10,15,20-tris-(tridecyl)-porphyrin (68 mg,4%), the second band the title compound5,15-bis-(3-hydroxyphenyl)-10,20-bis-(tridecyl)-porphyrin (52 mg, 2%)and the third band the title compound5,10-bis-(3-hydroxyphenyl)-15,20-bis-(tridecyl)-porphyrin (114 mg, 4%).

5,15-Bis-(3-hydroxyphenyl)-10,20-bis-(tridecyl)-porphyrin

violet microcrystalline solid, mp 133° C.; δ_(H)(500 MHz; CDCl₃) −2.70(2H, s, NH), 0.87 (6H, t, J 7.0, 2×CH₃), 1.25-1.33 (32H, m, 16×CH₂),1.46 (4H, m_(c), 2×CH₂), 1.72 (4H, m_(c), 2×CH₂), 2.46 (4H, m_(c),2×CH₂), 4.84 (4H, t, J 8.0, 2×CH₂), 7.15-7.17 (2H, Ar), 7.46-7.47 (2H,m, Ar), 7.54 (2H, d, J 7.5, Ar), 7.73 (2H, d, J 7.5, Ar), 8.83 (4H, d, J4.7, β-H), 9.33 (4H, d, J 4.7, β-H); δ_(C)(125 MHz; CDCl₃) 14.1 (CH₃),22.7 (CH₂), 29.3 (CH₂), 29.6 (CH₂), 29.7 (CH₂), 30.5 (CH₂), 31.9 (CH₂),35.2 (CH₂), 38.7 (CH₂), 114.6 (Ar), 118.2 (meso-C), 119.9 (meso-C),121.7 (Ar), 127.5 (Ar), 127.6 (β-C), 131.6 (β-C), 144.1 (Ar), 153.7(Ar); m/z (EI) 858 ([M]⁺, 100%), 689 ([M C₁₂H₁₅]⁺, 87), 429 ([M]²⁺, 9).

5,10-Bis-(3-hydroxyphenyl)-15,20-bis-(tridecyl)-porphyrin

violet microcrystalline solid, mp 113° C.; δ_(H)(500 MHz; CDCl₃) −2.68(2H, s, NH), 0.88 (6H, t, J 6.9, 2×CH₃), 1.27-1.37 (32H, m, 16×CH₂),1.46-1.52 (4H, m, 2×CH₂), 1.72-1.78 (4H, m, 2×CH₂), 2.46-2.52 (4H, m,2×CH₂), 4.86 (4H, t, J 7.0, 2×CH₂), 6.93-7.09 (2H, m, Ar), 7.25-7.30(2H, m, Ar), 7.38-7.46 (2H, m, Ar), 7.56-7.65 (2H, m, Ar), 8.54-8.62(2H, m, β-H), 8.75-8.79 (2H, m, β-H), 9.30 (2H, d, J 4.8, β-H) 9.49 (2H,s, β-H); δ_(C)(125 MHz; CDCl₃) 14.1 (CH₃), 22.7 (CH₂), 29.7 (CH₂), 30.6(CH₂), 31.9 (CH₂), 35.6 (CH₂), 38.9 (CH₂), 114.5 (Ar), 118.0 (mesa-C),120.3 (meso-C), 121.6 (Ar), 127.5 (Ar), 127.6 (Ar), 143.5 (Ar), 153.6(Ar); m/z (EI) 858 ([M]⁺, 100%), 689 ([M-C₁₂H₁₅]⁺, 36), 520 ([M-2C₁₂H₁₅]⁺, 6), 429 ([M]²⁺, 4).

1.3 Preparation of5,10,15-trihexyl-20-(4-methoxycarbonylphenyl)-porphyrin

In a typical experiment, dry dichloromethane (1500 ml) was placed in athree-necked flask equipped with a magnetic stirrer and argon gas inlet.After pyrrole (10.5 ml, 150 mmol), heptanal (15.8 ml, 113 mmol) andmethyl 4-formylbenzoate (6.2 g, 38 mmol) were added, the flask wasshielded from ambient light and TFA (2.15 ml, 28 mmol) was added and thereaction mixture was stirred at room temperature for 18 h. Then, DDQ (25g, 110 mmol) suspended in dry dichloromethane (100 ml) was added. Afterfurther stirring for 1 h, triethylarnine (6 ml) was added. To removepolymeric by-products, the reaction mixture was filtered through silicagel. The solvent was evaporated and separation was achieved via flashchromatography with dichloromethane and further purification withdichloromethane/hexane 3:1. Further purification was achieved byrecrystallization from dichloromethane/methanol The first band from thecolumn contained 5,10,15,20-tetrahexyl-porphyrin (930 mg, 5%), thesecond band the title compound5,10,15-trihexyl-20-(4-methoxycarbonylphenyl)-porphyrin (1400 mg, 5%).

5,10,15-Trihexyl-20-(4-methoxycarbonylphenyl)-porphyrin

violet microcrystalline solid; λ_(max)(CH₂Cl₂)/nm 418 (ε/dm³ mol⁻¹ cm⁻¹290300), 519 (14600), 553 (9100), 597 (4200) and 654 (6400); δ_(H)(250MHz; CDCl₃) −2.68 (2H, s, NH), 0.92-1.06 (9H, m, 3×CH₃), 1.33-1.60 (12H,m, 6×CH₂), 1.73-1.88 (6H, m, 3×CH₂), 2.43-2.60 (6H, m, 3×CH₂), 4.14 (3H,s, OCH₃), 4.85-4.96 (6H, m, CH₂), 8.25 (2H, d, J 8.2, Ar), 8.43 (2H, d,J 8.2, Ar), 8.73 (2H, d, J 5.5, β-H), 9.36 (2H, d, J 5.5, β-H), 9.45(2H, d, J 5.5, β-H), 9.48 (2H, d, J 5.5, β-H); δ_(C)(63 MHz; CDCl₃)14.30 (CH₃), 22.89 (CH₂), 22.93 (CH₂), 30.39 (CH₂), 30.46 (CH₂), 32.07(CH₂), 35.37 (CH₂), 35.96 (CH₂), 38.83 (CH₂), 39.04 (CH₂), 52.51 (OCH₃),116.54 (meso-C), 119.51 (meso-C), 120.01 (meso-C), 127.95 (Ar), 128.26(Ar), 129.52 (Ar), 130.84 (Ar), 134.63 (Ar), 147.77 (Ar), 167.58(CO₂CH₃); m/z (PSI) 697.4467 ([M+H]⁺ C₄₆H₅₇N₄O₂ ⁺ requires 697.4476).

Example 2 Preparation of Unsymmetrical Carboxy-Substituted Porphyrins2.1 Preparation of 5,15-bis-(4-carboxyphenyl)-10,20-dihexyl-porphyrin

In a typical experiment, a solution of KOH (200 mg, 3.6 mmol) inmethanol (1 ml) was added to a stirred solution of5,15-dihexyl-10,20-bis-(4-methoxycarbonylphenyl)-porphyrin (31 mg, 0.04mmol) in THF (8 ml) and the reaction mixture was stirred for 2 d. Water(50 ml) and hydrochloric acid were then added until the pH was adjustedto 4-6. The aqueous layer was extracted with dichloromethane (2×100 ml)and the organic layer was separated, washed with water until neutral anddried over sodium sulfate. The solvent was evaporated and the titlecompound 5,15-bis-(4-carboxyphenyl)-10,20-dihexyl-porphyrin was obtainedafter recrystallization from dichloromethane/aqueous methanol (26 mg,87%).

5,15-Bis-(4-carboxyphenyl)-10,20-dihexyl-porphyrin

violet microcrystalline solid, δ_(H)(250 MHz; (CD₃)₂SO) −2.96 (2H, s,NH), 0.81 (6H, t, J 7.1, 2×CH₃), 1.15-1.41 (8H, m, 4×CH₂), 1.60-1.71(4H, m, 2×CH₂), 2.25-2.37 (4H, m, 2×CH₂), 4.81-4.86 (4H, m, 2×CH₂), 8.21(4H, d, J 8.1, Ar), 8.33 (4H, d, J 8.1, Ar), 8.71 (4H, d, J 4.8, β-H),9.57 (4H, d, J 4.8, β-H); m/z (ESI) 719.3621 ([M+H]⁺, C₄₆H₄₇N₄O₄ ⁺requires 719.3592).

2.2 Preparation of 5,10-bis-(4-carboxyphenyl)-15,20-dihexyl-porphyrin

In a typical experiment, a solution of KOH (200 mg, 3.6 mmol) inmethanol (1 ml) was added to a stirred solution of5,10-dihexyl-15,20-bis-(4-methoxycarbonylphenyl)-porphyrin (34 mg, 0.05mmol) in THF (8 ml) and the reaction mixture was stirred for 2 d. Water(50 ml) and hydrochloric acid were then added until the pH was adjustedto 4-6. The aqueous layer was extracted with dichloromethane (2×100 ml)and the organic layer was separated, washed with water until neutral anddried over sodium sulfate. The solvent was evaporated and the titlecompound 5,10-bis-(4-carboxyphenyI)-15,20-dihexyl-porphyrin was obtainedafter recrystallization from dichloromethane/aqueous methanol (26 mg,79%).

5,10-bis-(4-carboxyphenyl)-15,20-dihexyl-porphyrin

violet microcrystalline solid, δ_(H)(250 MHz; (CD₃)₂SO) −2.99 (2H, s,NH), 0.81 (6H, t, J 7.0, 2×CH₃), 1.19-1.41 (8H, m, 4×CH₂), 1.60-1.71(4H, m, 2×CH₂), 2.25-2.36 (4H, m, 2×CH₂), 4.78-4.83 (4H, m, 2×CH₂), 8.18(4H, d, J 8.0, Ar), 8.30 (4H, d, J 8.0, Ar), 8.63 (2H, s, β-H), 8.69(2H, d, J 4.7, β-H), 9.54 (2H, d, J 4.7, β-H), 9.62 (2H, s, β-H); m/z(ESI) 719.3611 ([M +H]⁺, C₄₆H₄₇N₄O₄ ⁺ requires 719.3592).

Example 3 Preparation of Unsymmetrically Substitutedcis-dihydroxy-chlorins 3.1 Preparation of5,15-dihexyl-7,8-dihydroxy-10,20-bis-(4-methoxy-carbonylphenyl)-7,8-chlorin

In a typical experiment, a solution of osmium tetroxide (40 mg, 0.16mmol) in dichloromethane/pyridine 30% (4 ml) was added to a stirredsolution of 5,15-dihexyl-10,20-bis-(4-methoxycarbonylphenyl)-porphyrin(90 mg, 0.12 mmol) in dichloromethane/pyridine 30% (9 ml). Afterstirring for 3 h a saturated solution of sodium bisulfate inwater/methanol 1:1 (15 ml) was added and the mixture was stirred for 18h. The reaction mixture was filtered through Celite and dried oversodium sulfate. The solvent was evaporated and the residue was purifiedby flash chromatography with dichloromethane/ethylacetate 95:5 aseluent, followed by recrystallization from dichloromethane/methanol. Thefirst band from the column contained starting material (20 mg, 22%) andthe second band the title compound5,15-dihexyl-7,8-dihydroxy-10,20-bis-(4-methoxycarbonylphenyl)-7,8-chlorin(52 mg, 55%).

5,15-Dihexyl-7,8-dihydroxy-10,20-bis-(4-methoxycarbonylphenyl)-7,8-chlorin

violet microcrystalline solid, mp 124° C.; λ_(max)(CH₂Cl₂)/nm 411 (ε/dm³mol⁻¹ cm⁻¹ 182900), 428 (159800), 524 (15500), 550 (18400), 594 (8200)and 646 (19200); δ_(H)(500 MHz; CDCl₃) −1.94 (2H, br s, NH), 0.89-0.95(6H, m, 2×CH₃), 1.31-1.50 (8H, m, 4×CH₂), 1.67-1.76 (4H, m, 2×CH₂),2.15-2.29 (2H, m, CH₂), 2.33-2.41 (2H, m, CH₂), 4.08 (3H, s, OCH₃), 4.11(3H, s, OCH₃), 4.32-4.39 (1H, m, HCH), 4.48-4.54 (1H, m, HCH), 4.59-4.65(2H, m, CH₂), 6.22 (1H, d, J 7.1, β-H), 6.45 (1H, d, J 7.1, β-H), 7.95(1H, d, J 7.3, Ar), 8.04 (1H, d, J 7.4, Ar), 8.15 (2H, d, J 8.3, Ar),8.19 (1H, d, J 5.0, β-H), 8.32-8.39 (4H, br m, Ar), 8.42 (1H, d, J 4.6,β-H), 8.61 (1 H, d, J 4.9, β-H), 9.00 (1H, d, J 4.9, β-H), 9.06 (1H, d,J 4.6, β-H), 9.13 (1H, d, J 5.0, β-H); δ_(C)(125 MHz; CDCl₃) 14.26(CH₃), 14.30 (CH₃), 22.81 (CH₂), 22.91 (CH₂), 30.33 (CH₂), 30.42 (CH₂),31.98 (CH₂), 32.07 (CH₂), 33.38 (CH₂), 35.22 (CH₂), 36.76 (CH₂), 38.20(CH₂), 52.55 (OCH₃), 73.60 (β-C), 74.05 (β-C), 111.35 (meso-C), 113.39(meso-C), 120.54 (meso-C), 122.28 (β-C), 123.81 (meso-C), 124.14 (β-C),124.86 (β-C), 127.97 (Ar), 128.62 (β-C), 128.73 (Ar), 129.32 (Ar),129.65 (Ar), 129.75 (β-C), 129.81 (α-C), 132.35 (Ar), 133.09 (β-C),134.03 (Ar), 134.27 (Ar), 135.72 (α-C), 139.04 (α-C), 141.43 (α-C),146.84 (Ar), 147.29 (Ar), 151.59 (α-C), 153.67 (α-C), 159.05 (α-C),162.79 (α-C), 167.26 (CO₂CH₃), 167.50 (CO₂CH₃); m/z (ESI) 781.3988([M+H]⁺, C₄₈H₅₃N₄O₆ ⁺ requires 781.3960).

3.2 Preparation of5,10-dihexyl-7,8-dihydroxy-15,20-bis-(4-methoxy-carbonylphenyl)-7,8-chlorin,5,20-dihexyl-7,8-dihydroxy-10,15-bis-(4-methoxycarbonylphenyl)-7,8-chlorinand5,10-dihexyl-17,18-dihydroxy-15,20-bis-(4-methoxycarbonylphenyl)-17,18-chlorin

In a typical experiment, a solution of osmium tetroxide (100 mg, 0.39mmol) in dichloromethane/pyridine 30% (10 ml) was added to a stirredsolution of 5,10-dihexyl-15,20-bis-(4-methoxycarbonylphenyl)-porphyrin(226 mg, 0.30 mmol) in dichloromethane/pyridine 30% (35 ml). Afterstirring for 20 h a saturated solution of sodium bisulfate inwater/methanol 1:1 (40 ml) was added and the mixture was stirred for 18h. The reaction mixture was filtered through Celite and dried oversodium sulfate. The solvent was evaporated and the residue was purifiedby flash chromatography with dichloromethane/ethylacetate 90:10 aseluent, followed by recrystallization from dichloromethane/methanol. Thefirst band from the column contained starting material (38 mg, 18%), thesecond band the title compound5,10-dihexyl-7,8-dihydroxy-15,20-bis-(4-methoxycarbonylphenyl)-7,8-chlorin(60 mg, 25%), the third band the title compound5,20-dihexyl-7,8-dihydroxy-10,15-bis-(4-methoxycarbonylphenyl)-7,8-chlorin(74 mg, 31%) and the fourth band contained the title compound5,10-dihexyl-17,18-dihydroxy-15,20-bis-(4-methoxycarbonylphenyl)-17,18-chlorin(22 mg, 9%).

5,10-Dihexyl-7,8-dihydroxy-15,20-bis-(4-methoxycarbonylphenyl)-7,8-chlorin

violet microcrystalline solid, mp 171-178° C.; λ_(max)(CH₂Cl₂)/nm 410(ε/dm³ mol⁻¹ cm⁻¹ 346000), 426 (339000), 526 (31100), 553 (38900), 596(18100) and 648 (39400); δ_(H)(500 MHz; CDCl₃) −2.19 (2H, m, NH), 0.95(6H, t, J 7.3 Hz, 2×CH₃), 1.36-1.49 (8H, m, 4×CH₂), 1.66-1.75 (4H, m,2×CH₂), 2.09-2.23 (4H, m, 2×CH₂), 4.06 (6H, s, 2×OCH₃), 4.25-4.31 (2H,m, CH₂), 4.38-4.44 (2H, m, CH₂), 6.38 (2H, s, β-H), 7.90-7.98 (2H, br m,Ar), 8.02-8.09 (2H, br m, Ar), 8.14 (2H, br s, β-H), 8.28-8.30 (4H, m,Ar), 8.55 (2H, d, J 5.0, β-H), 8.95 (2H, d, J 5.0, β-H); δ_(C)(125 MHz;CDCl₃) 14.31 (CH₃), 22.93 (CH₂), 30.43 (CH₂), 32.04 (CH₂), 33.74 (CH₂),36.86 (CH₂), 52.52 (OCH₃), 73.89 (β-C), 113.47 (meso-C), 120.15(meso-C), 122.51 (β-C), 127.97 (β-C), 129.49 (β-C), 132.45 (Ar), 133.88(Ar), 134.19 (Ar), 134.69 (α-C), 140.16 (α-C), 146.83 (Ar), 152.33(α-C), 161.12 (α-C), 167.45 (CO₂CH₃); m/z (ESI) 781.3993 ([M+H]⁺,C₄₈H₅₃N₄O₆ ⁺ requires 781.3960).

5,20-Dihexyl-7,8-dihydroxy-10,15-bis-(4-methoxycarbonylphenyl)-7,8-chlorin

violet microcrystalline solid, mp 128° C.; λ_(max)(CH₂Cl₂)/nm 409 (ε/dm³mol⁻¹ cm⁻¹ 175100), 428 (143800), 524 (14900), 551 (17800), 594 (8400)and 647 (19800); δ_(H)(500 MHz; CDCl₃) −2.01 (1H, s, NH), −1.81 (1H, s,NH), 0.92-0.98 (6H, m, 2×CH₃), 1.34-1.52 (8H, m, 4×CH₂), 1.68-1.77 (4H,m, 2×CH₂), 2.12-2.20 (1H, m, HCH), 2.21-2.29 (1H, m, HCH), 2.34-2.40(2H, m, CH₂), 4.05 (3H, s, OCH₃), 4.09 (3H, s, OCH₃), 4.29-4.35 (1H, m,HCH), 4.42-4.49 (1H, m, HCH), 4.53 (2H, t, 18.3 Hz, CH₂), 6.15 (1H, d, J7.2, β-H), 6.37 (1H, d, J 7.2, β-H), 7.85 (1H, d, J 7.0, Ar), 7.95 (1H,d, J 7.0, Ar), 8.05-8.10 (1H, m, Ar), 8.11 (1H, d, J 4.9, β-H) 8.16-8.23(1H, m, Ar), 8.25-8.38 (4H, m, Ar), 8.42 (1H, d, J 4.6, β-H), 8.47 (1H,d, 4.9, β-H), 8.98 (1H, d, J 5.0, β-H), 9.04 (1H, d, 4.6, β-H), 9.16(1H, d, J 5.0, β-H); δ_(C)(125 MHz; CDCl₃) 14.28 (CH₃), 14.33 (CH₃),22.86 (CH₂), 22.95 (CH₂), 30.38 (CH₂), 30.46 (CH₂), 31.98 (CH₂), 32.09(CH₂), 33.68 (CH₂), 35.48 (CH₂), 36.80 (CH₂), 38.38 (CH₂), 52.52 (OCH₃),73.61 (β-C), 73.82 (β-C), 111.18 (meso-C), 113.40 (meso-C), 120.61(meso-C), 122.36 (β-C), 123.73 (meso-C), 123.95 (β-C), 125.72 (β-C),127.61 (β-C), 128.08 (Ar), 128.76 (Ar), 129.30 (Ar), 129.65 (Ar), 129.74(Ar), 130.02 (β-C), 132.24 (Ar), 132.71 (β-C), 133.95 (Ar), 134.16 (Ar),134.82 (α-C), 134.97 (α-C), 139.66 (α-C), 140.69 (α-C), 146.40 (Ar),146.90 (Ar), 151.92 (α-C), 153.22 (α-C), 159.91 (α-C), 161.68 (α-C),167.22 (CO₂CH₃), 167.46 (CO₂CH₃); m/z (EI) 780 ([M]⁺, 21%), 762 ([MH₂O], 61), 746 ([M-H₂OH]⁺, 71), 690 ([M-H₂O—C₅H₁₁]⁺, 51), 675([M-2OH—C₅H₁₁]⁺, 100), 604 ([M-2OH-2 C₅H₁₁]⁺, 30), 390 ([M]²⁺, 8), 381([M-H₂O]²⁺, 7), 373 ([M-2 OH]²⁺, 7); m/z HRMS (ET) 780.3889 ([M]^(+′),C₄₈H₅₂N₄O₆ ^(+′) requires 780.3881).

5,10-Dihexyl-17,18-dihydroxy-15,20-bis-(4-methoxycarbonylphenyl)-17,18-chlorin

violet microcrystalline solid, mp 121° C.; λ_(max)(CH₂Cl₂)/nm 409 (ε/dm³mol⁻¹ cm⁻¹ 163200), 428 (128300), 526sh (16300), 547 (19300), 593 (7700)and 645 (21000); δ_(H)(500 MHz; CDCl₃) −1.53 (2H, s, NH), 0.93 (6H, t, J7.3 Hz, 2×CH₃), 1.36-1.49 (8H, m, 4×CH₂), 1.73 (4H, m_(c), 2×CH₂), 2.40(4H, m_(c), CH₂), 4.05 (6H, s, 2×OCH₃), 4.58-4.67 (4H, m, 2×CH₂), 6.10(2H, s, β-H), 7.89 (2H, J 6.3, Ar), 8.08 (2H, J 6.3, Ar), 8.19 (2H, d, J5.0, β-H), 8.29 (2H, d, J 6.3, Ar), 8.35 (2H, d, J 6.3, Ar), 9.12 (2H,d, J 5.0 Hz, β-H), 9.13 (2H, s, β-H); δ_(C)(125 MHz; CDCl₃) 14.28 (CH₃),22.86 (CH₂), 30.37 (CH₂), 32.00 (CH₂), 35.56 (CH₂), 38.27 (CH₂), 52.47(OCH₃), 73.94 (β-C), 111.25 (meso-C), 123.83 (meso-C), 124.22 (β-C),125.41 (β-C), 128.67 (Ar), 129.14 (Ar), 129.63 (Ar), 130.34 (β-C),132.36 (Ar), 134.16 (Ar), 135.14 (α-C), 140.19 (α-C), 146.53 (Ar),152.94 (α-C), 160.57 (α-C), 167.31 (CO₂CH₃); m/z (ESI) 781.3989 ([M+H]⁺,C₄₈H₅₃N₄O₆ ⁺ requires 781.3960).

3.3 Preparation of7,8-dihydroxy-5,15-bis-(3-hydroxyphenyI)-10,20-bis-(trideeyl)-7,8-chlorin

In a typical experiment, a solution of osmium tetroxide (36 mg, 0.14mmol) in dichloromethane/pyridine 30% (4 ml) was added to a stirredsolution of 5,15-bis-(3-hydroxyphenyl)-10,20-bis-(tridecyl)-porphyrin(80 mg, 0.09 mmol) in dichloromethane/pyridine 30% (6 ml). Afterstirring for 5 h a saturated solution of sodium bisulfite inwater/methanol 1:1 (15 ml) was added and the mixture was stirred for 18h. The reaction mixture was filtered through Celite and dried oversodium sulfate. The solvent was evaporated and the residue was purifiedby flash chromatography with dichloromethane/ethylacetate 90:10 aseluent, followed by recrystallization from dichloromethane/aqueousmethanol. The first band from the column contained starting material (30mg, 39%) and the second band the title compound7,8-dihydroxy-5,15-bis-(3-hydroxyphenyl)-10,20-bis-(tridecyl)-7,8-chlorin(35 mg, 44%).

7,8-Dihydroxy-5,15-bis-(3-hydroxyphenyl)-10,20-bis-(tridecyl)-7,8-chlorin

violet microcrystalline solid, mp 111-120° C.; δ_(H)(500 MHz; CDCl₃)−1.88 (1H, s, NH), -1.65 (1H, s, NH), 0.82-0.85 (6H, m, 2×CH₃),1.22-1.34 (32H, m, 16×CH₂), 1.40-1.49 (4H, m, 2×CH₂), 1.67-1.74 (4H, m,2×CH₂), 2.22-2.40 (4H, m, 2×CH₂), 4.35-4.41 (1H, m, HCH), 4.61-4.71 (3H,m, CH₂, HCH), 6.26-6.33 (1H, m, β-H), 6.50-6.53 (1H, m, β-H), 7.16-7.18(1H, m, Ar), 7.25-7.28 (1H, m, Ar), 7.40-7.63 (6H, m, Ar), 8.35 (1H, d,J 4.8, β-H), 8.52 (1H, d, J 4.9, β-H), 8.75 (1H, d, 14.9, β-H), 9.14(2H, d, J 4.9, β-H), 9.32 (1H, d, J 4.8, β-H); δ_(C)(125 MHz; CDCl₃)13.5 (CH₃), 22.5 (CH₂), 28.9 (CH₂), 29.1 (CH₂), 29.2 (CH₂), 29.4 (CH₂),29.5 (CH₂), 29.6 (CH₂), 29.7 (CH₂), 30.2 (CH₂), 30.5 (CH₂), 31.8 (CH₂),34.5 (CH₂), 36.3 (CH₂), 38.1 (CH₂), 72.9 (β-C), 74.5 (β-C), 113.0(meso-C), 114.4 (Ar), 114.7 (Ar), 121.4 (Ar), 121.7 (α-C), 122.4 (Ar),124.1 (Ar), 124.4 (β-C), 125.8 (Ar), 127.6 (Ar), 128.3 (β-C), 129.2(β-C), 132.9 (β-C), 134.1 (α-C), 135.2 (α-C), 139.4 (α-C), 140.9 (α-C),143.8 (Ar), 151.8 (α-C), 153.4 (α-C), 155.8 (Ar); m/z (EI) 875([M-H₂O]^(+′), 37%), 856 ([M-2H₂O]^(+′), 13), 706 ([M-H₂O-C₁₂H₂₅]^(+′),6).

3.4 Preparation of17,18-dihydroxy-5,10-bis-(3-hydroxyphenyl)-15,20-bis-(tridecyl)-17,18-chlorin,7,8-dihydroxy-5,20-bis-(3-hydroxyphenyl)-10,15-bis-(tridecyl)-7,8-chlorinand7,8-dihydroxy-5,10-bis-(3-hydroxyphenyl)-15,20-bis-(tridecyl)-7,8-chlorin

In a typical experiment, a solution of osmium tetroxide (76 mg, 0.30mmol) in dichloromethane/pyridine 30% (8 ml) was added to a stirredsolution of 5,10-Bis-(3-hydroxyphenyl)-15,20-bis-(tridecyl)-porphyrin(185 mg, 0.22 mmol) in dichloromethane/pyridine 30% (10 ml). Afterstirring for 5 h a saturated solution of sodium bisulfite inwater/methanol 1:1 (20 ml) was added and the mixture was stirred for 18h. The reaction mixture was filtered through Celite and dried oversodium sulfate. The solvent was evaporated and the residue was purifiedby repeated flash chromatography with dichloromethane/ethylacetate90:10, 40:10 and dichloromethane/methanol 95:5 as eluent, followed byrecrystallization from dichloromethane/aqueous methanol. The first bandfrom the column contained starting material (19 mg, 10%), the secondband the title compound17,18-dihydroxy-5,10-bis-(3-hydroxyphenyl)-15,20-bis-(tridecyl)-17,18-chlorin(49 mg, 25%), the third band the title compound7,8-dihydroxy-5,20-bis-(3-hydroxyphenyl)-10,15-bis-(tridecyl)-7,8-chlorin(47 mg, 24%) and the fourth band contained the title compound7,8-dihydroxy-5,10-bis-(3-hydroxyphenyl)-15,20-bis-(tridecyl)-7,8-chlorin(25 mg, 13%).

17,18-Dihydroxy-5,10-bis-(3-hydroxyphenyl)-15,20-bis-(tridecyl)-17,18-chlorin

violet microcrystalline solid, mp 103° C.; δ_(H)(500 MHz; (CD₃)₂SO)−2.22 (2H, s, NH), 0.78 (6H, t, J 6.9 2×CH₃), 1.10-1.30 (32H, m,16×CH₂), 1.35-1.43 (4H, m, 2×CH₂), 1.60-1.70 (4H, m, 2×CH₂), 2.08-2.25(4H, m, 2×CH₂), 4.32-4.40 (2H, m, 2×HCH), 4.53-4.63 (2H, m, 2×HCH), 6.01(2H, s, β-OH), 6.44 (2H, s, β-H), 7.15-7.20 (2H, m, Ar), 7.45-7.54 (6H,m, Ar), 8.39 (2H, s, β-H), 8.72 (2H, d, J 4.7, β-H), 9.19 (2H, d, J 4.7,β-H), 9.79 (2H, s, Ar-OH); δ_(C)(125 MHz; (CD₃)₂SO) 14.4 (CH₃), 22.6(CH₂), 29.2 (CH₂), 29.5 (CH₂), 29.6 (CH₂), 30.5 (CH₂), 31.8 (CH₂), 33.4(CH₂), 36.6 (CH₂), 73.3 (β-C), 113.4 (meso-C), 115.4 (Ar), 120.8(meso-C), 122.9 β-C), 125.6 (Ar), 128.3 (Ar), 128.3 (β-C), 132.5 (β-C),134.5 (α-C), 140.0 (α-C), 143.1 (Ar), 152.1 (α-C), 156.4 (Ar), 164.0(α-C); m/z (EI) 892 ([M]^(+′), 5%), 874 ([M H₂O]^(+′), 100), 858 ([M-2OH)]^(+′), 31) 705 ([M-H₂O—C₁₂H₂₅]^(+′), 14).

7,8-Dihydroxy-5,20-bis-(3-hydroxyphenyl)-10,15-bis-(tridecyl)-7,8-chlorin

violet microcrystalline solid, mp 203-205° C.; δ_(H)(500 MHz; (CD₃)₂SO)−2.02 (1H, s, NH), −1.92 (1H, s, NH), 0.78-0.82 (6H, m, 2×CH₃),1.12-1.46 (36H, m, 18×CH₂), 1.59-1.71 (4H, m, 2×CH₂), 2.10-2.25 (2H, m,CH₂), 2.26-2.32 (2H, m, CH₂), 4.35-4.41 (1H, m, HCH), 4.60-4.66 (1H, m,HCH), 4.67-4.73 (2H, m, CH₂), 5.31 (1H, s, β-OH), 5.81 (1H, s, β-OH),6.05-6.11 (1H, m, β-H), 6.38-6.44 (1H, m, β-H), 7.05-7.07 (1H, m, Ar),7.14-7.19 (1H, m, Ar), 7.25-7.56 (6H, m, Ar), 8.23 (1H, d, J 4.8, β-H),8.43 (1H, d, J 4.6, β-H), 8.56 (1H, d, J 4.9, β-H), 9.17 (1H, β-H), 9.25(1H, d, J 4.8, β-H), 9.54 (1H, d, J 4.9, β-H); δ_(C)(125 MHz; (CD₃)₂SO)14.4 (CH₃), 14.5(CH₃), 22.6 (CH₂), 29.2 (CH₂), 29.5 (CH₂), 29.6 (CH₂),29.7 (CH₂), 31.8 (CH₂), 38.5 (CH₂), 73.3 (β-C), 74.6 (β-C), 122.8 (β-C),124.8 (β-C), 126.5 (β-C), 128.3 (β-C) 130.6 (β-C), 132.8 (β-C), 134.6(α-C), 143.1 (Ar), 151.7 (α-C), 152.6 (α-C), 164.1 (α-C); m/z (EI) 874([M-H₂O]^(+′), 20%), 705 ([M-H₂O—C₁₂H₂₅]^(+′), 9).

7,8-Dihydroxy-5,10-bis-(3-hydroxyphenyl)-15,20-bis-(tridecyl)-7,8-chlorin

violet microcrystalline solid, mp 137-141° C.; δ_(H)(500 MHz; (CD₃)₂CO)−1.56 (2H, s, NH), 0.83-0.86 (6H, m, 2×CH₃), 1.24-1.35 (32H, m, 16×CH₂),1.45-1.51 (4H, m, 2×CH₂), 1.72-1.78 (4H, m, 2×CH₂), 2.42 (4H, m_(c),2×CH₂), 4.70-4.80 (4H, m, 2×CH₂), 6.18-6.28 (2H, m, β-H), 7.12-7.15 (2H,m, Ar), 7.30-7.63 (6H, m, Ar), 8.37 (2H, d, J 4.8, β-H), 8.59 (2H, s,Ar—OH), 9.26 (2H, s, β-H), 9.36 (2H, J 4.8, β-H); δ_(C)(125 MHz;(CD₃)₂CO) 13.5 (CH₃), 22.5 (CH₂), 28.9 (CH₂), 29.1 (CH₂), 29.2 (CH₂),29.3 (CH₂), 29.4 (CH₂), 29.5 (CH₂), 29.6 (CH₂), 30.2 (CH₂), 31.8 (CH₂),34.9 (CH₂), 38.2 (CH₂), 73.9 (β-C), 112.7 (meso-C), 122.7 (meso-C),124.1 (β-C), 125.1 (β-C), 130.1 (β-C), 134.8 (α-C), 140.5 (α-C), 143.1(Ar), 152.6 (α-C), 162.6 (α-C); m/z (EI) 874 ([M-H₂O]^(+′), 1%), 705([M-H₂O—C₁₂H₂₅]^(+′), 1).

3.5 Preparation of5,10,15-trihexyl-7,8-dihydroxy-20-(4-methoxycarbonylphenyl)-7,8-chlorinand5,10,15-trihexyl-17,18-dihydroxy-20-(4-methoxycarbonylphenyl)-17,18-chlorin

In a typical experiment, a solution of osmium tetroxide (100 mg, 0.39mmol) in dichloromethane/pyridine 30% (10 ml) was added to a stirredsolution of 5,10,15-trihexyl-20-(4-methoxycarbonylphenyl)-porphyrin (212mg, 0.30 mmol) in dichloromethane/pyridine 30% (45 ml). After stirringfor 13 days a saturated solution of sodium bisulfite in water/methanol1:1 (40 ml) was added and the mixture was stirred for 18 h. The reactionmixture was filtered through Celite and dried over sodium sulfate. Thesolvent was evaporated and the residue was purified by repeated flashchromatography with dichloromethane/ethylacetate 95:5 as eluent,followed by recrystallization from dichloromethane/aqueous methanol. Thefirst band from the column contained starting material (103 mg, 49%),the second band the title compound5,10,15-trihexyl-7,8-dihydroxy-20-(4-methoxycarbonylphenyl)-7,8-chlorin(48 mg, 22%) and the third band the title compound5,10,15-trihexyl-17,18-dihydroxy-20-(4-methoxycarbonylphenyl)-17,18-chlorin(34 mg, 15%).

5,10,15-Trihexyl-7,8-dihydroxy-20-(4-methoxycarbonylphenyl)-7,8-chlorin

violet microcrystalline solid, mp 109-111° C.; λ_(max)(CH₂Cl₂)/nm 410(ε/dm³ mol⁻¹ cm⁻¹ 146100), 430 (121700), 527 (12400), 556 (16300), 596(7400), and 649 (14900); δ_(H)(500 MHz; CDCl₃) −2.15 (2H, br s, NH),0.90-0.94 (6H, m, 2×CH₃), 0.95 (3H, t, J 7.0, CH₃), 1.31-1.47 (12H, m,6×CH₂), 1.59-1.71 (6H, m, 3×CH₂), 1.97-2.12 (4H, m, 2×CH₂), 2.17-2.29(2H, m, CH₂), 4.02-4.37 (9H, m, 3×CH₂, OCH₃), 6.14-6.17 (2H, m, β-H),8.11-8.22 (2H, br m, Ar), 8.38 (2H, d, J 8.3 Hz, Ar), 8.42 (1H, d, J4.4, β-H), 8.59 (1H, d, J 4.8, β-H), 8.61 (1H, d, J 4.7, β-H), 8.73 (1H,d, J 4.7, β-H), 8.94 (1H, d, J 4.8, β-H), 8.99 (1H, d, J 4.4, β-H);δ_(C)(125 MHz; CDCl₃) 14.29 (CH₃), 14.33 (CH₃), 22.86 (CH₂), 22.89(CH₂), 22.94 (CH₂), 30.33 (CH₂), 30.34 (CH₂), 30.38 (CH₂), 31.95 (CH₂),32.01 (CH₂), 32.03 (CH₂), 33.24 (CH₂), 33.59 (CH₂), 36.57 (CH₂), 36.68(CH₂), 38.09 (CH₂), 52.56 (OCH₃), 73.33 (β-C), 73.83 (β-C), 112.20(meso-C), 112.24 (meso-C), 119.32 (meso-C), 121.83 (β-C), 122.24(meso-C), 122.43 (β-C), 124.87 (β-C), 127.97 (Ar), 128.01 (β-C), 129.52(Ar), 129.67 (β-C), 132.61 (β-C), 134.19 (Ar), 134.61 (α-C), 139.42(α-C), 140.38 (α-C), 147.34 (Ar), 151.53 (α-C), 152.94 (α-C), 159.70(α-C), 161.12 (α-C), 167.56 (CO₂CH₃); m/z (ESI) 731.4485 ([M+H]⁺C₄₆H₅₉N₄O₄ ⁺ requires 731.4531).

b5,10,15-Trihexyl-17,18-dihydroxy-20-(4-methoxycarbonylphenyl)-17,18-chlorin

violet microcrystalline solid; λ_(max)(CH₂CH₂)/nm 409 (ε/dm³ mol⁻¹ cm⁻¹209900), 430 (167300), 527 (17900), 554 (22200), 595 (10300) and 647(19800); δ_(H)(500 MHz; CDCl₃) −1.86 (2H, br s, NH), 0.92-0.97 (9H, m,3×CH₃), 1.34-1.51 (12H, m, 6×CH₂), 1.67-1.76 (6H, m, 3×CH₂), 2.07-2.29(2H, m, CH₂), 2.34-2.42 (4H, m, 2×CH₂), 4.06 (3H, s, OCH₃), 4.21-4.28(1H, m, HCH), 4.34-4.40 (1H, m, HCH), 4.52-4.59 (4H, m, 2×CH₂), 6.01(1H, d, J 7.2, β-H), 6.26 (1H, d, J 7.2, β-H), 7.79-7.90 (2H, br m, Ar),8.08 (1H, d, J 5.0, β-H), 8.23-8.33 (2H, br m, Ar), 8.91 (1H, d, J 5.0,β-H), 9.05 (1H, d, J 5.0, β-H), 9.09 (2H, s, β-H), 9.13 (1H, d, J 7.2,β-H); δ_(C)(125 MHz; CDCl₃) 14.30 (CH₃), 14.33 (CH₃), 22.87 (CH₂), 22.91(CH₂), 22.95 (CH₂), 30.37 (CH₂), 30.42 (CH₂), 32.00 (CH₂), 32.01 (CH₂),32.09 (CH₂), 33.34 (CH₂), 35.30 (CH₂), 35.58 (CH₂), 36.61 (CH₂), 38.15(CH₂), 38.33 (CH₂), 52.51 (OCH₃), 73.52 (β-C), 73.85 (β-C), 110.27(meso-C), 112.26 (meso-C), 121.92 (α-C), 122.91 (meso-C), 122.93(meso-C), 123.60 (β-C), 124.83 (β-C), 125.67 (β-C), 128.65 (Ar), 129.18(Ar), 129.54 (Ar), 129.92 (β-C), 130.21 (β-C), 132.34 (Ar), 133.90 (Ar),134.41 (α-C), 135.08 (α-C), 139.21 (α-C), 140.87 (α-C), 146.75 (Ar),152.35 (α-C), 152.76 (α-C), 158.99 (α-C), 161.59 (α-C), 167.30 (CO₂CH₃);m/z (EI) 730 ([M]⁺, 29%), 712 [M-H₂O]⁺, 100), 697 ([M-2OH]⁺, 42), 641([M-H₂O—C₅H₁₁]⁺, 57), 623 ([M-2OH—C₅H₁₁]⁺, 71); m/z HRMS (EI) 730.4454([M]^(+′), C₄₆H₅₈N₄O₄ ^(+′), requires 730.4453).

Example 4 Preparation of Unsymmetrical Carboxy-Substituted Chlorins 4.1Preparation of5,15-bis-(4-carboxyphenyl)-10,20-dihexyl-7,8-dihydroxy-7,8-chlorin

In a typical experiment, a solution of KOH (100 mg, 1.8 mmol) inmethanol (1 ml) was added to a stirred solution of5,15-dihexyl-7,8-dihydroxy-10,20-bis-(4-methoxycarbonylphenyl)-7,8-chlorin(20 mg, 0.016 mmol) in THF (3 ml) and the reaction mixture was stirredfor 2 d. Water (50 ml) and hydrochloric acid were then added until thepH was adjusted to 4-6. The aqueous layer was extracted withdichloromethane (2×100 ml) and the organic layer was separated, washedwith water until neutral and dried over sodium sulfate. The solvent wasevaporated and the title compound5,15-bis-(4-carboxyphenyl)-10,20-dihexyl-7,8-dihydroxy-7,8-chlorin wasobtained after recrystallization from dichloromethane/aqueous methanol(18 mg, 92%).

5,15-Bis-(4-carboxyphenyl)-10,20-dihexyl-7,8-dihydroxy-7,8-chlorin

violet microcrystalline solid, mp>300° C.; λ_(max)((CH₃)₂CO)/nm 407(ε/dm³ mol⁻¹ cm⁻¹ 154700), 427 (130300), 521 (13600), 548 (15300), 594(6500) and 646 (17600); δ_(H)(500 MHz; (CD₃)₂SO) −2.05 (1H, s, NH),−1.85 (1H, s, NH), 0.84 (3H, t, J 7.3 Hz, CH₃), 0.91 (3H, t, J 7.3,CH₃), 1.24-1.48 (8H, m, 4×CH₂), 1.64-1.67 (2H, m, CH₂), 1.69-1.75 (2H,m, CH₂), 2.12-2.23 (2H, m, CH₂), 2.25-2.31 (2H, m, CH₂), 4.35-4.41 (1H,m, HCH), 4.57-4.64 (1H, m, HCH), 4.66-4.72 (2H, m, CH₂), 5.40 (1H, br s,OH), 5.86 (1H, br s, OH), 6.09 (1H, d, J 6.9, β-H), 6.40 (1H, d, J 6.9,β-H), 8.02-8.35 (10H, m, 8×Ar, 2×β-H), 8.64 (1H, d, J 5.0, β-H), 9.19(1H, d, J 4.7, β-H), 9.24 (1H, d, J 5.0, β-H), 9.45 (1H, d, J 5.2, β-H);δ_(C)(125 MHz; (CD₃)₂SO) 13.93 (CH₃), 14.02 (CH₃), 22.10 (CH₂), 22.23(CH₂), 29.30 (CH₂), 29.67 (CH₂), 31.27 (CH₂), 31.36 (CH₂), 33.88 (CH₂),111.99 (meso-C), 113.10 (meso-C), 119.32 (meso-C), 122.64 (β-C), 123.87(β-C), 127.79 (Ar), 128.23 (β-C), 132.40 (β-C), 133.59 (Ar), 146.10(Ar), 146.46 (Ar) 167.50 (CO₂H), 167.67 (CO₂H); m/z (ESI) 753.3664([M+H]⁺, C₄₆H₄₉N₄O₆ ⁺ requires 753.3647).

4.2 Preparation of5,20-bis-(4-carboxyphenyl)-10,15-dihexyl-7,8-dihydroxy-7,8-chlorin

In a typical experiment, a solution of KOH (200 mg, 3.6 mmol) inmethanol (1 ml) was added to a stirred solution of5,20-dihexyl-7,8-dihydroxy-10,15-bis-(4-methoxycarbonylphenyl)-7,8-chlorin(35 mg, 0.045 mmol) in THF (3 ml) and the reaction mixture was stirredfor 2 d. Water (50 ml) and hydrochloric acid were then added until thepH was adjusted to 4-6. The aqueous layer was extracted withdichloromethane (2×100 ml) and the organic layer was separated, washedwith water until neutral and dried over sodium sulfate. The solvent wasevaporated and the title compound5,20-bis-(4-carboxyphenyl)-10,15-dihexyl-7,8-dihydroxy-7,8-chlorin wasobtained after recrystallization from dichloromethane/aqueous methanol(31 mg, 91%).

5,20-Bis-(4-carboxyphenyl)-10,15-dihexyl-7,8-dihydroxy-7,8-chlorin

violet microcrystalline solid, mp>300° C.; λ_(max)((CH₃)₂CO)/nm 407(ε/dm³ mol⁻¹ cm⁻¹ 122800), 427 (96000), 523 (10100), 548 (11500), 595(4600) and 646 (13900); δ_(H)(500 MHz; (CD₃)₂CO) −1.88 (1H, s, NH),−1.74 (1H, s, NH), 0.91 (3H, t, J 7.3 Hz, CH₃), 0.96 (3H, t, J 7.3,CH₃), 1.34-1.56 (8H, m, 4×CH₂), 1.75-1.85 (4H, m, 2×CH₂), 2.28-2.46 (4H,m, 2×CH₂), 4.44-4.50 (1H, m, HCH), 4.65-4.72 (1H, m, HCH), 4.77 (2H,m_(c), CH₂), 6.33 (1H, d, J 7.0, β-H), 6.53 (1H, d, J 7.0, β-H), 8.24(1H, d, J 4.9, β-H), 8.00-8.40 (8H, br m, Ar), 8.42 (1H, d, J 4.5, β-H),8.56 (1H, d, J 4.9, β-H), 9.21 (1H, d, J 4.5, β-H), 9.30 (1H, d, J 5.0,β-H), 9.56 (1H, d, J 5.0, β-H); δ_(C)(125 MHz; (CD₃)₂SO) 14.00 (CH₃),14.10 (CH₃), 22.19 (CH₂), 22.33 (CH₂), 29.38 (CH₂), 29.76 (CH₂), 31.33(CH₂), 31.44 (CH₂), 32.86 (CH₂), 34.39 (CH₂), 36.16 (CH₂), 38.12 (CH₂),72.91 (β-C), 73.13 (β-C), 111.93 (meso-C), 113.17 (meso-C), 119.45(meso-C), 122.66 (β-C), 124.10 (β-C), 126.43 (β-C), 127.44 (β-C), 127.92(Ar), 129.52 (Ar), 130.20 (β-C), 132.11 (β-C), 133.76 (α-C), 133.87(Ar), 134.29 (α-C), 139.06 (α-C), 140.04 (α-C), 145.84 (Ar), 146.22(Ar), 150.78 (α-C), 152.25 (α-C), 163.07 (α-C), 163.91 (α-C), 167.53(CO₂H), 167.71 (CO₂H); m/z (ESI) 753.3637 ([M+H]⁺, C₄₆H₄₉N₄O₆ requires753.3647).

4.3 Preparation of5-(4-carboxyphenyl)-10,15,20-trihexyl-17,18-dihydroxy-17,18-chlorin

In a typical experiment, a solution of KOH (100 mg, 1.8 mmol) inmethanol (0.5 ml) was added to a stirred solution of5,10,15-trihexyl-7,8-dihydroxy-20-(4-methoxycarbonylphenyl)-7,8-chlorin(12 mg, 0.016 mmol) in THF (2 ml) and the reaction mixture was stirredfor 2 d. Water (50 ml) and hydrochloric acid were then added until thepH was adjusted to 4-6. The aqueous layer was extracted withdichloromethane (2×100 ml) and the organic layer was separated, washedwith water until neutral and dried over sodium sulfate. The solvent wasevaporated and the title compound5-(4-carboxyphenyl)-10,15,20-trihexyl-17,18-dihydroxy-17,18-chlorin wasobtained after recrystallization from dichloromethane/aqueous methanol(10 mg, 87%).

5-(4-Carboxyphenyl)-10,15,20-trihexyl-17,18-dihydroxy-17,18-chlorin

violet microcrystalline solid, mp 134° C.; λ_(max)((CH₃)₂CO)/nm 408(ε/dm³ mol⁻¹ cm⁻¹ 88600), 428 (73300), 526 (8500), 553 (9200), 595(4300) and 649 (7400); δ_(H)(500 MHz; (CD₃)₂CO) −1.98 (1H, s, NH), −1.96(1H, s, NH), 0.88-0.98 (9H, m, 3×CH₃), 1.33-1.55 (12H, m, 6×CH₂),1.73-1.85 (6H, m, 3×CH₂), 2.20-2.45 (6H, m, 3×CH₂), 4.38-4.47 (2H, m,CH₂), 4.60-4.67 (2H, m, CH₂), 4.75 (2H, m_(c), CH₂), 5.30 (1H, br s,COOH), 6.57 (2H, s, β-H), 8.19 (2H, d, J 8.1 Hz, Ar), 8.38 (1H, d, J4.5, β-H), 8.41 (2H, d, J 8.1, Ar), 8.64 (1H, d, J 4.9, β-H), 9.16-9.18(2H, m, β-H), 9.24 (1H, d, J 5.0, β-H), 9.49 (1H, d, J 5.0, β-H);δ_(C)(125 MHz; (CD₃)₂CO) 14.37 (CH₃), 14.44 (CH₃), 14.47 (CH₃), 23.40(CH₂), 23.50 (CH₂), 23.54 (CH₂), 30.77 (CH₂), 31.04 (CH₂), 31.09 (CH₂),32.64, (CH₂), 32.71 (CH₂), 32.74 (CH₂), 34.12 (CH₂), 34.49 (CH₂), 35.54(CH₂), 37.24 (CH₂), 37.37 (CH₂), 39.06 (CH₂), 74.14 (β-C), 74.28 (β-C),113.62 (meso-C), 113.70 (meso-C), 119.70 (meso-C), 122.71 (meso-C),123.05 (β-C), 123.13 (β-C), 126.16 (β-C), 128.42 (β-C), 128.88 (Ar),130.50 (β-C), 130.79 (α-C), 133.08 (β-C), 134.83 (Ar), 135.70 (α-C),140.53 (α-C), 141.03 (α-C), 148.04 (Ar), 152.14 (α-C), 153.83 (α-C),163.12 (α-C), 164.13 (α-C), 167.87 (CO₂H); m/z (ESI) 717.4399 ([MH]⁺C₄₅H₅₇N₄O₄ ⁺ requires 717.4374).

4.4 Preparation of5-(4-carboxyphenyl)-10,15,20-trihexyl-7,8-dihydroxy-7,8-chlorin

In a typical experiment, a solution of KOH (200 mg, 3.6 mmol) inmethanol (1 ml) was added to a stirred solution of5,10,15-trihexyl-17,18-dihydroxy-20-(4-methoxycarbonylphenyl)-17,18-chlorin(25 mg, 0.034 mmol) in THF (3 ml) and the reaction mixture was stirredfor 2 d. Water (50 ml) and hydrochloric acid were then added until thepH was adjusted to 4-6. The aqueous layer was extracted withdichloromethane (2×100 ml) and the organic layer was separated, washedwith water until neutral and dried over sodium sulfate. The solvent wasevaporated and the title compound5-(4-carboxyphenyl)-10,15,20-trihexyl-7,8-dihydroxy-7,8-chlorin wasobtained after recrystallization from dichloromethane/aqueous methanol(22 mg, 90%).

5-(4-Carboxyphenyl)-10,15,20-trihexyl-7,8-dihydroxy-7,8-chlorin

violet microcrystalline solid, mp 128-133° C.; λ_(max)((CH₃)₂CO)/nm 404(ε/dm³ mol⁻¹ cm⁻¹ 152200), 428 (114600), 525 (13300), 550 (15900), 595(6600) and 647 (18200); δ_(H)(500 MHz; (CD₃)₂CO) −1.78 (1H, s, NH),−1.59 (1H, s, NH), 0.87-0.96 (9H, m, 3×CH₃), 1.30-1.53 (12H, m, 6×CH₂),1.68-1.80 (6H, m, 3×CH₂), 2.18-2.27 (1H, m, HCH), 2.29-2.42 (5H, m,2×CH₂, HCH), 4.32-4.82 (1H, m, HCH), 4.54-4.60 (1H, m, HCH), 4.61-4.72(4H, m, 2×CH₂), 6.17 (1H, d, J 7.1 β-H), 6.40 (1H, d, J 7.1 β-H),7.89-7.95 (1H, br m, Ar), 8.11-8.16 (1H, m, Ar), 8.17 (1H, d, J 4.9,β-H), 8.22-8.36 (2H, br m, Ar), 9.14 (1H, d, J 5.0, β-H), 9.16 (1H,β-H), 9.18 (1H, d, J 4.7, β-H), 9.26 (1H, d, J 4.9, β-H), 9.41 (1H, d, J5.0, β-H); δ_(C)(125 MHz; (CD₃)₂CO) 14.37 (CH₃), 14.40 (CH₃), 14.46(CH₃), 23.38 (CH₂), 23.42 (CH₂), 23.51 (CH₂), 30.73 (CH₂), 30.79 (CH₂),31.05 (CH₂), 32.62, (CH₂), 32.63 (CH₂), 32.72 (CH₂), 33.93 (CH₂), 35.48(CH₂), 35.82 (CH₂), 37.16 (CH₂), 38.92 (CH₂), 39.12 (CH₂), 73.88 (β-C),74.55 (β-C), 112.13 (mesa-C), 113.27 (meso-C), 122.71 (β-C), 122.93(meso-C), 122.96 (meso-C), 124.48 (β-C), 125.68 (β-C), 126.56 (β-C),130.22 (α-C), 130.67 (β-C), 130.98 (β-C), 135.07 (α-C), 135.70 (α-C),140.12 (α-C), 141.49 (α-C), 148.03 (Ar), 153.09 (α-C), 153.50 (α-C),162.07 (α-C), 164.25 (α-C), 168.01 (CO₂H); m/z (ESI) 717.4356 ([M+H]⁺C₄₅H₅₇N₄O₄ ⁺ requires 717.4374).

Example 5 Preparation of Porphyrins Containing Carbohydrate Moieties 5Preparation of5,15-dihexyl-10,20-bis-(3-β-D-acetoglucosylphenyl)-porphyrin and5,10-dihexyl-15,20-bis-(3-β-D-acetoglucosylphenyl)-porphyrin

In a typical experiment, dry dichloromethane (528 ml) was placed in athree-necked flask equipped with a magnetic stirrer and argon gas inlet.After pyrrole (528 μl, 7.62 mmol), heptanal (560 μl, 3.96 mmol) andtetraacetyl-β-D-glucopyranosyloxy-benzaldehyde (600 mg, 1.32 mmol) wereadded, the flask was shielded from ambient light and TFA (408 μl, 5.28mmol) was added and the reaction mixture was stirred at room temperaturefor 18 h. Then, DDQ (898 mg, 3.96 mmol) suspended in dry dichloromethane(30 ml) was added. After further stirring for 1 h, triethylamine (1 ml)was added. To remove polymeric by-products, the reaction mixture wasfiltered through silica gel. The solvent was evaporated and purificationwas achieved via repeated flash chromatography with hexane/ethylacetate60:40 and dichloromethane/ethylacetate 90:10 as eluent, followed byrecrystallization from dichloromethane/methanol The first band from thecolumn contained 5,10,15,20-tetrahexyl-porphyrin (51 mg, 8%), the secondband 5,10,15-trihexyl-20-bis-(3-β-D-acetoglucosylphenyl)-porphyrin (145mg, 11%), the third band the title compound5,15-dihexyl-10,20-bis-(3-β-D-acetoglucosylphenyl)-porphyrin (33 mg, 4%)and the fourth band contained the title compound5,10-dihexyl-15,20-bis-(3-β-D-acetoglucosylphenyl)-porphyrin (57 mg,7%).

5,15-Dihexyl-10,29-bis-(3-β-D-acetoglucosylphenyl)-porphyrin

violet microcrystalline solid, δ_(H)(500 MHz; CDCl₃) −2.72 (2H, s, NH),0.91-0.96 (6H, m, 2×CH₃), 1.31-1.33 (6H, m, Ac), 1.36-1.43 (4H, m,2×CH₂), 1.48-1.56 (4H, m, 2×CH₂), 1.77-1.84 (4H, m, 2×CH₂), 1.98 (6H, s,Ac), 2.04 (6H, s, Ac), 2.11 (6H, s, Ac), 2.49-2.55 (4H, m, 2×CH₂),3.76-3.81 (2H, m, H-5 ‘ose’), 4.03-4.07 (2H, m, H-6 ‘ose’) 4.14-4.18(2H, m, H-6 ‘ose’), 4.94-5.00 (4H, m, 2×CH₂), 5.15-5.20 (2H, m, H-4‘ose’), 5.31-5.34 (2H, m, H-2/3 ‘ose’), 5.35-5.37 (2H, m, H-1 ‘ose’),5.39-5.42 (2H, m, H-2/3 ‘ose’), 7.43-7.46 (2H, m, Ar), 7.66-7.69 (2H, m,Ar), 7.84-7.87 (2H, m, Ar), 7.92-7.95 (2H, m, Ar), 8.89 (4H, d, J 4.7,β-H), 9.43-9.46 (4H, m, β-H); δ_(C)(125 MHz; CDCl₃) 14.23 (CH₃), 19.96(COCH₃), 20.60 (COCH₃), 20.69 (COCH₃), 20.81 (COCH₃), 22.82 (CH₂), 30.31(CH₂), 31.99 (CH₂), 35.42 (CH₂), 38.88 (CH₂), 61.99 (C-6 ‘ose’), 68.38(C-4 ‘ose’), 71.40 (C-2/3 ‘ose’), 72.26 (C-5 ‘ose’), 72.89 (C-2/3‘ose’), 99.38 (C-1 ‘ose’), 116.71 (Ar), 118.10 (meso-C), 120.21(meso-C), 122.77 (Ar). 127.67 (Ar), 128.07 (β-C), 129.94 (Ar), 131.70(β-C), 144.31 (Ar), 155.34 (Ar), 169.44 (2×COCH₃), 170.30 (COCH₃),170.45 (COCH₃); m/z (ESI) 1323.5 ([M+H]⁺ C₇₂H₈₃N₄O₂₀ ⁺ requires 1323.6).

5,10-Dihexyl-15,20-bis-(3-β-D-acetoglucosylphenyl)-porphyrin

violet microcrystalline solid, δ_(H)(500 MHz; CDCl₃) −2.71 (2H, s, NH),0.95-0.98 (6H, m, 2×CH₃), 1.32-1.38 (6H, m, Ac), 1.40-1.47 (4H, m,2×CH₂), 1.52-1.59 (4H, m, 2×CH₂), 1.82-1.89 (4H, m, 2×CH₂), 1.98-2.01(6H, m, Ac), 2.04-2.05 (6H, m, Ac), 2.10-2.12 (6H, m, Ac), 2.52-2.61(4H, m, 2×CH₂), 3.75-3.84 (2H, m, H-5 ‘ose’), 4.02-4.07 (2H, m, H-6‘ose’) 4.14-4.21 (2H, m, H-6 ‘ose’), 5.00-5.04 (4H, m, 2×CH₂), 5.15-5.21(2H, m, H-4 ‘ose’), 5.30-5.43 (6H, m, H-1, H-2, H-3 ‘ose’), 7.41-7.45(2H, m, Ar), 7.63-7.70 (2H, m, Ar), 7.82-7.95 (4H, m, Ar), 8.76-8.79(2H, m, β-H), 8.87-8.91 (2H, m, β-H) 9.45-9.49 (2H, m, β-H), 9.59 (2H,s, β-H); δ_(C)(125 MHz; CDCl₃) 14.25 (CH₃), 19.96 (COCH₃), 20.61(COCH₃), 20.69 (COCH₃), 20.80 (COCH₃), 22.85 (CH₂), 30.41 (CH₂), 32.02(CH₂), 35.84 (CH₂), 39.05 (CH₂), 62.01 (C-6 ‘ose’), 68.39 (C-4 ‘ose’),71.39 (C-2/3 ‘ose’), 72.27 (C-5 ‘ose’), 72.89 (C-2/3 ‘ose’), 99.24 (C-1‘ose’), 116.52 (Ar), 117.75 (meso-C), 120.68 (meso-C), 122.79 (Ar),129.95 (Ar), 129.94 (Ar), 143.97 (Ar), 155.45 (Ar), 169.45 (2×COCH₃),170.31 (COCH₃), 170.47 (COCH₃); (ESI) 1323.5 ([M+H]⁺C₇₂H₈₃N₄O₂₀ ⁺requires 1323.6).

Example 6 Cell Tests of Selected Compounds in the HT 29 Cell Line

The photosensitzing activity was determined in the human colonadenocarcinoma cell line HT29. The HT29 cell lines were grown in DMEM(cc-pro GmbH) supplemented with 10% heat-inactivated fetal calf serum(FCS, cc-pro GmbH), 1% penicillin (10000 IU) and streptomycin (10000μg/ml, cc-pro GmbH). Cells were kept as a monolayer culture in ahumidified incubator (5% CO₂ in air at 37° C.

A photosensitizer stock solution (2 mM) was performed in DMSO and waskept in the dark at 4° C. Further dilution was performed in RPMI 1640medium without phenol red supplemented with 10% FCS to reach a finalphotosensitizer concentration of 2 or 10 μM, respectively.

2·10⁴ cells/ml were seeded in micro plates (2·10⁵ cells/well). Cellswere incubated with fresh medium (RPMI without phenol red) containing10% FCS with 2 or 10 μM of the photosensitizer for 24 h before lightexposure. Before photosensitization, cells were washed, incubated withRPMI without phenol red and 10% FCS, then irridiated at room temperaturewith a 652 nm diode laser (Ceralas PDT 652, biolitec AG) at a fixedfluence rate of 100 mW/cm² (50 J/cm²). Following irradiation, cells wereincubated in a humidified incubator (5% CO₂ in air at 37° C.) for 24 huntil cell viability assay.

The cell viability was assessed by the XTT assay. 500 mg XTT (sodium3′-[phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzenesulfonic acid, Applichem GmbH) is dissolved in 500 ml PBS-Buffer(without Ca²⁺ and Mg²) and sterile filtered. Solution was stored in thedark at −20° C. until use. A sterile solution containing PMS (N-methyldibenzopyrazine methyl sulfate, Applichem GmbH) was needed as anactivation reagent for the XTT. 0.383 mg PMS was dissolved in 1 mlPBS-Buffer The solution should be stored frozen and should not beexposed to light. The XTT reagent solution was thawed in a 37° C. waterbath and the activation solution (PMS) was added immediately prior touse. To prepare a reaction solution sufficient for one micro plate (96wells), 0.1 ml activation solution (PMS) was given to 5 ml XTT reagent.The medium in the micro plate was exchanged with fresh RPM, withoutphenol red and 10% FCS (100 μl) prior adding 50 μl XTT reaction solutionper well. The micro plate was incubated for 2-3 hours at 37° C. and 5%CO₂ until an orange dye is to be formed. The micro plate has been shakengently to evenly distribute the dye in the wells.

The absorbance of the samples was measured with a spectrophotometer(Bio-Kinetics Reader EL312 e; Bio-Tek Instruments Inc.) at a wavelengthof 490 nm. In order to measure reference absorbance (to measurenon-specific readings) a wavelength of 630-690 nm was used.

The examples 6.1 to 6.3 illustrate the photodynamic activity (DT meansdark toxicity and Laser means photo toxicity) of photosensitizers havinga substitution pattern as referred to in the present invention. Thethree photosensitizers have an A₂B₂-substitution pattern of themeso-substituents, combining two polar and two nonpolarmeso-substituents. Especially5,15-bis-(4-carboxyphenyl)-10,20-dihexyl-7,8-dihydroxy-7,8-chlorinexhibits a very strong photodynamic activity in the HT29 cell line,which is known to be very resistant against cell-toxic agents and PDT aswell.

The examples 6.4 to 6.6 are included to illustrate, that chlorinphotosensitizers which do not have a substitution pattern as referred toin the present invention do not exhibit a promising photodynamicactivity in the cell experiments. The three photosensitizers in thiscase are based on an A₃B-substitution pattern of the meso-substituents.In fact, in example 6.6, where there are three hexyl chains and onebenzoic ester group at the meso-positions the cell viability seems toincrease (!) under irradiation (for a photosensitizer concentration of10 μM, cf. below).

Example 7 Cell Tests of Selected Compounds in a Rabbit Synoviocyte and aMouse Macrophage Cell Line, HIG82 and J774A.1

The mouse monocytes-macrophages cell line J774A.1 and the rabbitsynoviocyte cell line HIG-82 were grown in DMEM (cc-pro GmbH)supplemented with 10% heat-inactivated fetal calf serum (FCS, cc-proGmbH), 1% penicillin (10000 IU) and streptomycin (10 000 μg/ml, cc-proGmbH). Cells were kept as a monolayer culture in a humidified incubator(5% CO₂ in air at 37° C.).

A photosensitizer stock solution (2 mM) was performed in DMSO and waskept in the dark at 4° C. Further dilution was performed in RPMI 1640medium without phenol red supplemented with 10% FCS to reach a finalphotosensitizer concentration of 2 or 10 μM, respectively.

2·10⁴ cells/ml were seeded in micro plates (2·10⁵ cells/well). Cellswere incubated with fresh medium (RPMI without phenol red) containing10% FCS with 2 or 10 μM of the photosensitizer for 24 h before lightexposure. Before photosensitization, cells were washed, incubated withRPMI without phenol red and 10% FCS, then irridiated at room temperaturewith a 652 nm diode laser (Ceralas PDT 652, biolitec AG) at a fixedfluence rate of 100 mW/cm² (50 J/cm²). Following irradiation, cells wereincubated in a humidified incubator (5% CO₂ in air at 37° C.) for 24 huntil cell viability assay.

The cell viability was assessed by the XTT assay. 500 mg XTT (sodium3′-[phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzenesulfonic acid, Applichem GmbH) is dissolved in 500 ml PBS-Buffer(without Ca²⁺ and Mg²) and sterile filtered. Solution was stored in thedark at −20° C. until use. A sterile solution containing PMS (N-methyldibenzopyrazine methyl sulfate, Applichem GmbH) was needed as anactivation reagent for the XTT. 0.383 mg PMS was dissolved in 1 mlPBS-Buffer The solution should be stored frozen and should not beexposed to light. The XTT reagent solution was thawed in a 37° C. waterbath and the activation solution (PMS) was added immediately prior touse. To prepare a reaction solution sufficient for one micro plate (96wells), 0.1 ml activation solution (PMS) was given to 5 ml XTT reagent.The medium in the micro plate was exchanged with fresh RPMI withoutphenol red and 10% FCS (100 μl) prior adding 50 μl XTT reaction solutionper well. The micro plate was incubated for 2-3 hours at 37° C. and 5%CO₂ until an orange dye is to be formed. The micro plate has been shakengently to evenly distribute the dye in the wells.

The absorbance of the samples was measured with a spectrophotometer(Bio-Kinetics Reader EL312 e; Bio-Tek Instruments Inc.) at a wavelengthof 490 nm. In order to measure reference absorbance (to measurenon-specific readings) a wavelength of 630-690 nm was used.

The examples 7.1 to 7.4 illustrate the photodynamic activity ofphotosensitizers having a substitution pattern as referred to in thepresent invention against synoviocytes and macrophages, cell types whichare especially relevant for the treatment of arthritis and similarinflammatory diseases. The four photosensitizers have theA₂B₂-substitution pattern of the meso-substituents, combining two polarand two nonpolar meso-substituents. Especially,5,15-bis-(4-carboxyphenyl)-10,20-dihexyl-7,8-dihydroxy-7,8-chlorin and5,20-bis-(4-carboxyphenyl)-10,15-dihexyl-7,8-dihydroxy-7,8-chlorinexhibit a very strong photodynamic activity in the two cell lines.

The example 7.5 is again included to illustrate, that chlorinphotosensitizers which do not have a substitution pattern as referred toin the present invention do not exhibit a promising photodynamicactivity.

Example 8 Cell Testing of a Liposomal Formulation of5,15-bis-(4-carboxyphenyl)-10,20-dihexyl-7,8-dihydroxy-7,8-chlorin

The liposomal formulation of the photosensitizer5,15-bis-(4-carboxyphenyl)-10,20-dihexyl-7,8-dihydroxy-7,8-chlorin wasprepared in analogy to the procedure described in the U.S. Pat. No.7,354,599 B2 by V. Albrecht, A. Fahr et al.

The tests with the liposomal formulation illustrate that thephotosensitizers of the present invention may be formulated intoliposomes for the purpose of e.g. a PDT or arthritis treatment withoutlosing their photodynamic activity.

The liposomal formulation of the photosensitizer can be used to e.g.influence the pharmacokinetics of photosensitizer absorption anddistribution and increase the bioavailability.

Having described preferred embodiments of the invention with referenceto the accompanying examples, it is to be understood that the inventionis not limited to the precise embodiments, and that various changes andmodifications may be effected therein by skilled in the art withoutdeparting from the scope of the invention as defined in the appendedclaims.

1-24. (canceled)
 25. An unsymmetrically meso-substituted tetrapyrroliccompound having the formula:

wherein B is selected from the group consisting of:

wherein any two of R, (called R¹) are the same or different substitutedor unsubstituted alkyl groups or fluoroalkyl groups consisting of 4-15carbon atoms; and wherein the remaining two R are phenyl groups havingthe formula:

where R² is a substituent either in the meta- or para-position of thephenyl ring and R² is selected from the group consisting of OH, COOH,NH₂, COOX, NHX, OX, NH—Y—COOH and CO—Y—NH₂ , where X is a polyethyleneglycol residue with (CH₂CH₂O)_(n)CH₃ with n=1-30 or a carbohydratemoiety and Y is a peptide or oligopeptide wherein n=1-30.
 26. Thetetrapyrrolic compound according to claim 25 based specifically on theformulas 1, 2, 3 or 4:

Wherein R¹ is: a substituted or unsubstituted alkyl group or fluoroalkylgroup consisting of 4-15 carbon atoms, and Wherein B, R² are defined asin claim
 25. 27. The tetrapyrrolic compound according to claim 26 basedon the formulas 1, 2, 3 or 4:

Wherein: B is selected from the group consisting of:

and R¹, R² are as defined in claim
 26. 28. The tetrapyrrolic compoundaccording to claim 27 specifically based on formula 1:


29. The tetrapyrrolic compound according to claim 27, based on formula1:

Wherein: B is defined as in claim 27, or a pharmaceutically acceptablederivative thereof.
 30. A method of photodynamic therapy comprisingadministering to a patient a predetermined amount of a compound of claim25 or a pharmaceutically acceptable derivative thereof, pausing apredetermined time and exposing said patient to light of a predeterminedintensity and wavelength.
 31. A method for diagnosing or treatingarthritis or inflammatory diseases comprising administering to a patientan effective amount of the compound of claim 25 or a pharmaceuticallyacceptable derivative thereof.
 32. A pharmaceutical compositioncomprising a compound according to claim 25 or a pharmaceuticallyacceptable derivative thereof as an active ingredient.
 33. Thepharmaceutical composition according to claim 32 wherein said compoundor said pharmaceutically acceptable derivative thereof is conjugated toa targeting agent.
 34. The pharmaceutical composition according to claim33 in which said targeting agent is selected from a group consisting ofan antibody, a fragment of an antibody and a peptide.
 35. Thepharmaceutical composition according to claim 32 in which saidpharmaceutical composition is a liposomal formulation.
 36. Thepharmaceutical composition according to claim 33 in which saidpharmaceutical composition is a liposomal formulation.
 37. A method ofproducing compounds of claim 25, wherein substituents on a parentporphyrin (tetrapyrrole) are preselected to direct reduction ordihydroxylation to form a certain isomer of a corresponding chlorin. 38.The method of production according to claim 37, wherein saidsubstituents are selected by their steric and/or electronic influence todirect dihydroxylation or reduction with diimine to a favored isomer.39. The method according to claim 37, wherein in an intermediate step anosmate(VI)ester is reductively cleaved without use of gaseous H₂S.